Postsynaptic assembly

Acetylcholine reception

The response to acetylcholine is mediated via multiple types of ionotropic nicotinic ACh receptors (for review see (Brown, 2006), G-protein-linked muscarinic receptors (Lee et al., 1999b; Lee et al., 2000; Min et al., 2000), and ACh-gated chloride channels (Putrenko et al., 2005).

The muscarinic-type ACh receptors in C. elegans are encoded by 1, 2, and gar-3 genes. The primary transcripts from all three gar genes undergo alternative splicing leading to a large protein diversity (Park et al., 2003; Park et al., 2000; Suh et al., 2001). Analyses of GFP constructs expression indicate that gar-1 and gar-2 are expressed in neurons (Lee et al., 2000) while gar-3 is expressed in pharyngeal muscle (Steger and Avery, 2004).

The ACh-gated chloride channel (ACC) subunits in C. elegans belong to the superfamily of Cys loop ligand-gated ion channels. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. Two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. All these four ACC proteins have no orthologs in vertebrates or Drosophila (Putrenko et al., 2005). Nothing has yet been reported about the expression patterns of these proteins, or mutant phenotypes.

The “standard” vertebrate nicotinic type of AChR is a pentameric ligand-gated anion channel. Analysis of the C. elegans genome has revealed one of the most-extensive and diverse nAChR gene families known, consisting of at least 27 subunits (reviewed in (Jones and Sattelle, 2004; Schafer, 2002). Based on sequence similarity, these protein subunits can be classified in five classes: UNC-29, UNC-38, ACR-16, ACR-8, and DEG-3 (Jones and Sattelle, 2004; Mongan et al., 1998; Mongan et al., 2002). The last two classes represent nematode specific groups. Each class contains three to nine members, and some of the classes contain both " and non-" type subunits. Some nAChR subunits are particular to neurons whilst others are present in both neurons and muscles. The muscles of the body wall express two major types of ACh receptors (Richmond and Jorgensen, 1999):

• levamisole-sensitive receptors, heteromeric, containing three essential subunits: UNC-29, UNC-38, and UNC-63 (Culetto et al., 2004; Fleming et al., 1997). Another two subunits LEV-1 and LEV-8 are present in a subset of the receptors and seem non-essential for the response to levamisole (Towers et al., 2005).

• nicotine-sensitive receptors, homomeric, containing only the ACR-16 " subunit (Francis et al., 2005; Touroutine et al., 2005).

Eliminating the function of either the levamisole-sensitive receptor or the nicotine-sensitive receptor induces none or mild uncoordinated phenotype, while eliminating the function of both receptors (e.g., unc-29; acr-16 or unc-63;acr-16 double mutant) leads to a synthetic severe uncoordinated phenotype (Francis et al., 2005; Touroutine et al., 2005).

Several proteins are associated with cholinergic function and are required for the expression, maturation, trafficking, and/or localization of particular ACh receptor subunits.

RIC-3 is required for the maturation of at least four types of nAChR: EAT-2, DEG-3/DES-2, UNC-29 and ACR-16 (Halevi et al., 2002). RIC-3 is localized to cell bodies and is expressed in almost all neurons and in the muscles of body wall and pharynx. ric-3 mutants are uncoordinated, small, slow growing, resistant to cholinesterase inhibitors and levamisole (Miller et al., 1996; Nguyen et al., 1995).

UNC-50 is a membrane protein that localizes to the Golgi apparatus and is required in body wall muscles for the trafficking of the assembled Lev-AChR to the NMJ. In unc-50 mutants, the Lev-AChR is rapidly degraded by the lysosomal system after receptor assembly (Eimer et al., 2007).

LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for ionotropic AChR clustering (e.g. UNC-29). In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, while the number of functional AChRs present at the muscle cell surface remains unchanged (Gally et al., 2004).

LEV-9 is a component of extracellular scaffold required for both L-AChRs and LEV-10 clustering at the NMJs. In lev-9 mutants, L-AChRs are still present at the muscle cell surface, but their concentration is strongly reduced at the synapse. Localization of both 9 and LEV-10 at the synapse requires the function of both proteins (Marie Gendrel, personal communication).

EAT-18 is required for proper function of pharyngeal EAT-2 non-" subunit, and apparently affects most or all of the nicotinic receptors in the pharynx. eat-18 mutants, like eat-2 mutants, are incapable of rapid pharyngeal pumping (McKay et al., 2004). Surprisingly, the eat-18 gene and promoter are nested within the first intron of lev-10 (Gally et al., 2004).

CAM-1 is a Ror receptor tyrosine kinase required for the localization and clustering of ACR-16 (Francis et al., 2005). CAM-1 is present in many neurons, including ventral cord cholinergic motor neurons, and in muscles, being enriched in the muscle arms and especially at NMJs. Analysis of cam-1 mutants shows that the protein is also involved in cell migration and axon guidance (Forrester et al., 1999).

There are also some proteins that clearly contribute to the stability, trafficking, and/or clustering of AChR subunits and whose loss-of-function phenotypes are associated with partial reduction in the abundance of localized receptors.

Biogenic amine reception

Seventeen putative biogenic amine receptors have been identified based on sequence similarity to mammalian receptors and many of their mutants are available in C. elegans (Chase and Koelle, 2007).

Four dopamine receptors (DOP-1 to 4) have been identified in C. elegans. Mutants in dop-3 fail to slow in response to a bacterial lawn, defect rescued by mutations in dop-1 suggesting that DOP-1 and DOP-3 antagonize each other to control the rate of locomotion in response to changes in the environmental stimuli (Chase et al., 2004). Mutations in the dop-1 receptor also cause defects in the ability of the animals to respond to mechanical stimulation (plate tapping). DOP-1 functions in the touch neurons to modulate their response. DOP-2 has only recently been recognized as a dopamine receptor in C. elegans (Suo et al., 2003) and because of its expression in dopaminergic neurons, this receptor may function as an autoreceptor and regulate the synthesis and the release of dopamine (Suo et al., 2004).

Furthermore, four receptors have been identified for serotonin, including one serotonin-gated chloride channel (MOD-1) and three G protein coupled metabotropic receptors (SER-1, SER-4 and SER-7). MOD-1 is required for the enhanced slowing response (Ranganathan et al., 2000; Sawin et al., 2000). SER-1 is expressed on the vulval muscles and ser-1 mutants fail to lay eggs in response to serotonin. SER-1 is additionally expressed on

pharyngeal muscles and may be responsible for the observed effect of serotonin on the repolarization of the pharyngeal muscles (Carnell et al., 2005; Dempsey et al., 2005; Niacaris and Avery, 2003). SER-4 and SER-7 are also expressed in pharyngeal neurons or muscles and so might also play a role in controlling pharyngeal activity (Hobson et al., 2006; Tsalik et al., 2003).

SER-3 appears to be an octopamine receptor and may be activated by octopamine in the SIA interneurons in response to starvation (Suo et al., 2006).

Two receptors, SER-2 and TYRA-2, bind tyramine with relatively high affinity when expressed in cell culture (Rex et al., 2005; Rex and Komuniecki, 2002). ser-2 mutants fail to suppress head oscillations in response to touch, similar to the behavioural defects seen in tdc-1 (which encodes the enzyme required for tyramine synthesis) mutants (Rex et al., 2004).

Behavioural analysis of TYRA-2 mutant has not yet been published.

GABA reception

There are two GABA receptors described in C. elegans: a GABAA-gated chloride channels encoded by the unc-49 gene and a GABA-gated cation channel encoded by exp-1 gene. unc-49 encodes three distinct GABA receptor subunits by splicing a common N-terminal ligand-binding domain to one of three alternative C-terminal domains, producing the UNC-49A, UNC-49B, and UNC-49C subunits. GABAA receptors are involved in the inhibitory GABA neurotransmission, thus UNC-49 inhibits body muscle contraction during locomotion (Bamber et al., 1999; Bamber et al., 2005; Gally and Bessereau, 2003). exp-1 mutants exhibit defects in excitatory GABA functions but are normal for the inhibitory GABA functions (Beg and Jorgensen, 2003). Specifically, exp-1 mutants lack enteric muscle contractions, but move and forage normally (McIntire et al., 1993b).

Glutamate reception

In C. elegans, 10 putative ionotropic glutamate receptor (iGluRs) subunits have been identified: two NMDA (encoded by nmr-1 and nmr-2 genes), and eight non-NMDA receptor subunits (GLR-1 to GLR-8) most similar to either the AMPA or kainate subfamilies (Brockie et al., 2001). Also, glutamate-gated chloride channels (GluCl), encoded by glc-1, glc-2, glc-3, glc-4, avr-14/gbr-2 and avr-15, were identified in pharmacological screens (using the anthelmintic drug avermectin)(Cully et al., 1994; Dent et al., 1997; Dent et al., 2000; Horoszok et al., 2001;

Vassilatis et al., 1997). GluCls are expressed in both pharyngeal muscle cells and neurons and they are involved in pharyngeal pumping, sensory perception and locomotion (Dent et al., 2000).

iGluRs are ligand-gated ion channels formed by the heteromeric assembly of four receptor subunits from a single class (NMDA or non-NMDA), reviewed in (Brockie and Maricq, 2003). The distribution of all known C. elegans iGluRs was described using GFP fusion molecules (Brockie et al., 2001; Maricq et al., 1995). While some subunits are widely expressed throughout the nervous system, others are expressed only in a single neuronal pair. For example, GLR-3 and GLR-6 subunits can be found only in RIA neurons, which are part of a neuronal circuit required for worm thermotactic behaviour, suggesting that GLR-3 and GLR-6 are required for thermotaxis (Mori and Ohshima, 1995). The interneurons of the locomotion control circuit (AVA, AVB, AVD, AVE and PVC) express up to four non-NMDA subunits (GLR-1, GLR-2, GLR-4 and GLR-5) and the two NMDA subunits (NMR-1 and NMR-2) suggesting that glutamate reception plays important roles in the control of worm locomotion. GLR-7 and GLR-8 are expressed in the pharyngeal nervous system (Brockie et al., 2001).

Recent studies have emphasized that distinct mechanisms control synaptic expression of the two classes NMDA and AMPA receptors (reviewed by (Bredt and Nicoll, 2003). NMDA receptor proteins are relatively fixed, whereas AMPA receptors rapidly cycles between a subcellular pool and the postsynaptic membrane. A large family of interacting proteins regulates the AMPA receptor turnover at synapses and thereby influences synaptic strength.

ire-1 and xbp-1 are proteins involved in the unfolded protein response (UPR), regulating the export of assembled iGluR subunits from the endoplasmic reticulum (ER).

Mutations of these genes prevent GLR-1, GLR-2, and GLR-5 exports from the ER (Shim et al., 2004). Also, mutations in the pore domain – modifying ion permeability, and mutations in the ligand-binding domain – disrupting glutamate binding, dramatically reduced the synaptic abundance of GLR-1 and increased retention of GLR-1 in the ER (Grunwald and Kaplan, 2003).

lin-10, which encodes a PZD-domain protein, is required for proper localization and stabilization of iGluR at the postsynaptic membrane. Mutation in lin-10 change the normal punctate distribution of GLR-1::GFP (synaptic) to an uniform distribution (Rongo et al., 1998).

CDK5, a proline-directed kinase, phosphorylates LIN-10 and regulates LIN-10 and GLR-1 abundance in the ventral cord, suggesting that phosphorilation of LIN-10 may play a role in the anterograde trafficking of GLR-1 (Glodowski et al., 2007).

unc-42, which encodes calcium and calmodulin-dependent protein kinase II (CaMKII), is also required for proper synaptic localization of GLR-1. Loss-of-function mutation in unc-43 disrupts trafficking, leading to the accumulation of GLR-1 in the cell bodies and, subsequently, a reduction in the density of GLR-1 synapses (Rongo and Kaplan, 1999).

sol-1 encodes a CUB-domain transmembrane protein that is required either for the gating open of GLR-1 receptors or for their rate of desensitization after opening (Walker et al., 2006b). However, the mechanism of SOL-1 function is not elucidated. sol-1 mutants show the

same behavioural defects as glr-1 mutants and furthermore the GLR-1-dependent glutamate-gated current is absent (Brockie and Maricq, 2006; Zheng et al., 2006; Zheng et al., 2004).

rab-10 encodes a small GTPase required for GLR-1 trafficking and recycling using a cholesterol-dependent endocytosis pathway. Mutations in rab-10 lead to intracellular endosomal accumulation of GLR-1 in cell bodies (Glodowski et al., 2007).

unc-11, ortholog of the AP180 clathrin adaptor protein, is also involved in the internalization of GLR-1 and regulation of the GluR postsynaptic density. Disrupting unc-11 leads to increased levels of GLR-1 at postsynaptic elements in ventral nerve cord (Burbea et al., 2002), suggesting that clathrin-mediated endocytosis is required for compensatory regulation of GLR-1 after activity blockade (Grunwald et al., 2004).

GLR-1 was found to be ubiquitinated in vivo and overexpressing ubiquitin in GLR-1::GFP transgenic worms leads to the reduction of both density and amplitude of Glu-GLR-1::GFP puncta. Moreover, GLR-1::GFP accumulates in the ventral cord when the fusion protein has specific mutations that blocks ubiquitination (Burbea et al., 2002). All these results indicate that ubiquitin-mediated degradation of GLR-1 contributes to the regulation of the synaptic pools and, thus, modulating the strength of glutamatergic signalling.

APC (anaphase-promoting complex) regulates also the abundance of GLR-1 at synapses (Juo and Kaplan, 2004). Mutations in the genes that encode the subunits of the APC result in the accumulation of GLR-1 at ventral cord synapses. APC is a multisubunit ubiquitin ligase that assembles ubiquitin chains on substrate molecules thereby regulating their degradation. One of the best-known and most studied targets of APC is !-catenin.

LIN-23-mediated degradation of !-catenin regulates also the abundance of GLR-1 in the ventral nerve cord. lin-23 encodes a subunit of SCF (Skp1/Cullin/F-box) ubiquitin ligase, which modulates the !-catenin/Wnt pathway to alter postsynaptic GLR-1 levels (Dreier et al., 2005).

KEL-8 (kelch-repeat containing protein 8) binds to CUL-3, a Cullin 3 ubiquitin ligase, and both are required for the ubiquitin-mediated turnover of GLR-1. kel-8 mutants show an increased frequency of spontaneous reversals in locomotion, indicating increased levels of synaptic GLR-1 (Schaefer and Rongo, 2006).

The regulation of synaptic GLR-1 density might be used to maintain plasticity of the nervous system by either strengthening or weakening synaptic connections. One way to realize that is by ubiquitination of GLR-1, which enables clathrin-mediated endocytosis of GLR-1 via an UNC-11-dependent mechanism.