Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in <em>Caenorhabditis elegans</em>

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the ...

Okinawa Institute of Science and Technology Graduate University

Neurotrophins (NTs) and their receptors play crucial roles in the development, functions and maintenance of nervous systems. It is widely believed that NT-induced dimerization of the receptors initiates the transmembrane signaling. However, it is still controversial whether the receptor molecule has a monomeric or dimeric structure on the cell surface before its ligand binding.

Brain-derived neurotrophic factor receptor TrkB exists as a preformed dimer in living cells.

Survival requires that living organisms continuously monitor environmental and tissue pH. Animals sense acidic pH using ion channels and G-protein-coupled receptors (GPCRs), but monitoring of alkaline pH is not well understood. We report here that in the nematode Caenorhabditis elegans, a transmembrane receptor-type guanylyl cyclase (RGC), GCY-14, of the ASEL gustatory neuron, plays an essential role in the sensing of extracellular alkalinity. Activation of GCY-14 opens a cGMP-gated cation channel encoded by tax-2 and tax-4, resulting in Ca(2+) entry into ASEL. Ectopic expression of GCY-14 in other neurons indicates that it accounts for the alkalinity sensing capability. Domain-swapping and site-directed mutagenesis of GCY-14 reveal that GCY-14 functions as a homodimer, in which histidine of the extracellular domains plays a crucial role in alkalinity detection. These results argue that in addition to ion channels and GPCRs, RGCs also play a role in pH sensation in neurons.

Environmental alkalinity sensing mediated by the transmembrane guanylyl cyclase GCY-14 in C. elegans.

Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. 

Mechanisms of activation of receptor tyrosine kinases: monomers or dimers.

The cell-surface receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). To understand the molecular mechanisms underlying the induction of Tar activity by its ligands, the Escherichia coli Tar periplasmic domain with and without bound aspartate (Asp-Tar and apo-Tar, respectively) were each crystallized in two different forms. Using ammonium sulfate as a precipitant, crystals of apo-Tar1 and Asp-Tar1 were grown and diffracted to resolutions of 2.10 and 2.40 Å, respectively. Alternatively, using sodium chloride as a precipitant, crystals of apo-Tar2 and Asp-Tar2 were grown and diffracted to resolutions of 1.95 and 1.58 Å, respectively. Crystals of apo-Tar1 and Asp-Tar1 adopted space group P41212, while those of apo-Tar2 and Asp-Tar2 adopted space groups P212121 and C2, respectively. 

Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate receptor Tar and its complex with aspartate.

Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range.

Alkaline pH sensor molecules.

It has long been thought that transmembrane cell-surface receptors, such as receptor tyrosine kinases and cytokine receptors, among others, are activated by ligand binding through ligand-induced dimerization of the receptors. However, there is growing evidence that prior to ligand binding, various transmembrane receptors have a preformed, yet inactive, dimeric structure on the cell surface. Various studies also demonstrate that during transmembrane signaling, ligand binding to the extracellular domain of receptor dimers induces a rotation of transmembrane domains, followed by rearrangement and/or activation of intracellular domains. The paper here describes transmembrane cell-surface receptors that are known or proposed to exist in dimeric form prior to ligand binding, and discusses how these preformed dimers are activated by ligand binding. 

Activation of transmembrane cell-surface receptors via a common mechanism? The "rotation model".

Invertebrate models have generated many new insights into transmembrane signaling by cell-surface receptors. This review focuses on receptor guanylyl cyclases (rGCs) and describes recent advances in understanding their roles in sensory processing in the nematode, . A complete analysis of the  genome elucidated 27 rGCs, an unusually large number compared with mammalian genomes, which encode 7 rGCs. Most  rGCs are expressed in sensory neurons and play roles in sensory processing, including gustation, thermosensation, olfaction, and phototransduction, among others. Recent studies have found that by producing a second messenger, guanosine 3',5'-cyclic monophosphate, some rGCs act as direct sensor molecules for ions and temperatures, while others relay signals from G protein-coupled receptors. Interestingly, genetic and biochemical analyses of rGCs provide the first example of an obligate heterodimeric rGC. Based on recent structural studies of rGCs in mammals and other organisms, molecular mechanisms underlying activation of rGCs are also discussed in this review.

Receptor Guanylyl Cyclases in Sensory Processing.

The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer's disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA). We demonstrated that exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains, to form a complex that localizes mainly to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex was inhibited by p75NTR, ShcC and Mint-2. Importantly, we demonstrated that the association between endogenous APP and TrkA in primary septal neurons were modified by NGF, or by drugs that either inhibit ER-to-Golgi transport or perturb microtubules and microfilaments. Interestingly, several agents that induce cell death [amyloid β (Aβ)-peptide, staurosporine and rapamycin], albeit via different mechanisms, all caused dissociation of APP/TrkA complexes and increased production of C-terminal fragment (β-CTF) APP fragment. These findings open new perspectives for investigating the interplay between these proteins during neurodegeneration and AD.

Association of TrkA and APP Is Promoted by NGF and Reduced by Cell Death-Promoting Agents.

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility, and differentiation. The dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for -autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in the reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the "rotation model"). This model is also able to explain how oncogenic mutations activate the receptor in the absence of the ligand, without assuming that the mutations induce receptor dimerization. In this review, we discuss the mechanisms underlying the ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model.

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The "Rotation Model".

Active propagation of electrical signals in C. elegans neurons requires ion channels capable of regenerating membrane potentials. Here we report regenerative depolarization of a major gustatory sensory neuron, ASEL. Whole-cell patch-clamp recordings in vivo showed supralinear depolarization of ASEL upon current injection. Furthermore, stimulation of animal's nose with NaCl evoked all-or-none membrane depolarization in ASEL. Mutant analysis showed that EGL-19, the α1 subunit of L-type voltage-gated Ca channels, is essential for regenerative depolarization of ASEL. ASEL-specific knock-down of EGL-19 by RNAi demonstrated that EGL-19 functions in C. elegans chemotaxis along an NaCl gradient. These results demonstrate that a natural substance induces regenerative all-or-none electrical signals in dendrites, and that these signals are essential for activation of sensory neurons for chemotaxis. As in other vertebrate and invertebrate nervous systems, active information processing in dendrites occurs in C. elegans, and is necessary for adaptive behavior.

Active propagation of dendritic electrical signals in C. elegans.

Two people with the same lifespan do not necessarily have the same healthspan. One person may retain locomotor and cognitive abilities until the end of life, while another person may lose them during adulthood. Unbiased searches for genes that are required to maintain locomotor ability during adulthood may uncover key regulators of locomotor healthspan. Here, we take advantage of the relatively short lifespan of the nematode  and develop a novel screening procedure to collect mutants with locomotor deficits that become apparent in adulthood. After ethyl methanesulfonate mutagenesis, we isolated five  mutant strains that progressively lose adult locomotor ability. In one of the mutant strains, a nonsense mutation in , which encodes Elongator Complex Protein Component 2, causes a progressive decline in locomotor ability during adulthood. Mutants and mutations identified in the present screen may provide insights into mechanisms of age-related locomotor impairment and the maintenance of locomotor healthspan.

Forward Genetic Screen for  Mutants with a Shortened Locomotor Healthspan.

Some genes are essential for survival, while other genes play modulatory roles on health and survival. Genes that play modulatory roles may promote an organism's survival and health by fine-tuning physiological processes. An unbiased search for genes that alter an organism's ability to maintain aspects of health may uncover modulators of lifespan and healthspan. From an unbiased screen for  mutants that show a progressive decline in motility, we aimed to identify genes that play a modulatory role in maintenance of locomotor healthspan. Here we report the involvement of  encoding a class I histone deacetylase, as a genetic factor that contributes in the maintenance of general health and locomotion in . We identified a missense mutation in HDA-3 as the causative mutation in one of the isolated strains that show a progressive decline in maximum velocity and travel distance. From transcriptome analysis, we found a cluster of genes on Chromosome II carrying BATH domains that were downregulated by . Furthermore, downregulation of individual  genes leads to significant decline in motility. Our study identifies genetic factors that modulate the maintenance of locomotor healthspan and may reveal potential targets for delaying age-related locomotor decline.

Mutation in histone deacetylase HDA-3 leads to shortened locomotor healthspan in .

The nematode  is widely used for behavioral studies ranging from simple chemosensation to associative learning and memory. It is vital for such studies to determine optimal concentrations of attractive and aversive chemicals that  can sense. Here we describe a resource localization assay in which a chemical compound of interest is placed in two compartments of a quadrant plate in order to determine optimal concentrations of the chemical in behavioral studies. Using the assay, we determined the optimal concentration of a water-soluble attractant, KCl, as an unconditioned stimulus for the study of associative learning and memory. In this protocol, we also describe a chemotaxis assay using a square agar plate spotted with an aversive olfactory cue, 1-nonanol, as a conditioned stimulus.

Ichiro N. Maruyama

Information Processing Biology Unit

Okinawa Institute of Science and Technology Graduate University

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

Ichiro N. Maruyama

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Information Processing Biology Unit

Okinawa Institute of Science and Technology Graduate University

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

Information Processing Biology Unit