Why Study Glutamate Receptors?

Neuronal communication is the primary means by which our nervous system senses, interprets, remembers, and responds to the outside world and to our own internal physiology. Much of this communication occurs at chemical synapses, which are specialized signaling structures comprised of a presynaptic cell that releases neurotransmitters, and a postsynaptic cell that detects these neurotransmitters using receptor proteins.

Our research is focused on glutamate receptors (GluRs), which detect glutamate, the major excitatory neurotransmitter in our brain. We are particularly interested in how GluRs are localized to synapses because such glutamate receptor cell biology plays an important role in synaptic communication, synaptic plasticity, and learning and memory. In addition, glutamate receptors are implicated in several diseases of the nervous system, and are a primary neurodegenerative agent activated by mechanical damage (e.g., traumatic injury) and by oxygen deprivation (e.g., stroke). Thus, a better understanding of these receptors will facilitate the diagnosis, treatment, and prevention of diseases attributable to neurodegeneration, and help us better understand the mechanisms behind learning and memory.

Our focus has been to identify the factors that regulate GluR localization and function using a genetic approach in the nematode C. elegans. We use C. elegans because its simple nervous system, which is easily-visualized through its transparent body (see below; image from Dr. Yishi Jin), allows us to observe glutamate receptor trafficking within neurons in an intact and behaving animal. My lab has used the rich genetic and genomic tools of this organism, and both forward and reverse genetic approaches, to identify multiple genes that function in glutamate receptor biology. All of the genes we have identified have human equivalents that seem to be playing similar or identical roles in the human brain, suggesting that our findings are likely to be applicable to human health.

GLR-1, a C. elegans Glutamate Receptor Subunit

In the mid 1990’s, papers from several labs showed that PDZ-domain proteins bind to GluRs, and that GluRs could be differentially sorted in primary neuronal cultures. In addition, researchers began using GFP to study protein localization, particularly in C. elegans. Consequently, Dr. Rongo decided to study protein trafficking in the C. elegans nervous system. He joined the lab of Dr. Josh Kaplan, who, in collaboration with Dr. Cori Bargmann’s group, had recently identified GLR-1, a C. elegans AMPA-type glutamate receptor, based on the behavioral phenotype of glr-1 mutants.

GLR-1 functions in a simple touch circuit. C. elegans can sense touch at the tip of their nose via a pair of mechanosensory neurons, called ASH. ASH makes synaptic connections on a group of “command” interneurons, which regulate whether worms move forwards or backwards. In response to touch at the tip of the animal’s nose, wild-type worms activate an escape response by reversing locomotion to avoid the stimulus. Loss-of-function mutants for glr-1 fail to respond to touch. By contrast, gain-of-function mutations in glr-1 (e.g., the lurcher mouse mutation introduced into glr-1 by Dr. Villu Maricq’s lab) result in worms that spontaneously reverse even in the absence of a stimulus. Thus, GLR-1 activity, which functions in the command interneurons that receive sensory neuron input, regulates when worms reverse locomotion.

Dr. Rongo reasoned that GLR-1 would be postsynaptically localized; thus, he generated transgenic nematodes that express chimeric GLR-1::GFP, and found that GLR-1::GFP is localized to synaptic connections. Using this transgene, he looked for candidate genes that were required for proper GLR-1 localization. Through these studies, he found that the PDZ-domain protein LIN-10, originally identified by Dr. Stuart Kim for its role in epithelial polarity, is required to localize GLR-1 to postsynaptic clusters. This work provided some of the early genetic evidence that PDZ-domain proteins play a role in glutamate receptor localization to neuronal synapses. Through similar approaches, Dr. Rongo also found that worm CaMKII, identified as UNC-43 by Dr. Jim Thomas, regulates GluR trafficking in C. elegans neurons. This work provided the first genetic evidence that CaMKII signaling regulates glutamate receptor trafficking, and complemented ongoing electrophysiological studies by Dr. Roberto Malinow indicating a similar role for CaMKII in mammalian neurons. Taken together, these two studies provided a new cell biological and molecular explanation for how CaMKII might facilitate learning and memory by mobilizing intracellular pools of GluRs in response to synaptic activity.

Dr. Rongo’s Research Program At Rutgers

Forward Genetic Screens to Study GluR Localization.

When Dr. Rongo established his lab at Rutgers in 2000, glutamate receptor localization and function remained a large and important problem, and most of the underlying molecules remained unknown. The lab took a new approach by doing forward genetic screens to identify new genes that regulate GLR-1 localization. Such forward genetic screens identify genes solely based on their function, and thus lack any preconceived bias with respect to the underlying molecules. Instead of relying on behavioral screens, we chose to screen for GLR-1::GFP localization mutants by directly screening through mutant nematodes by epifluorescence microscopy. While genetic screens can be somewhat risky, our screen has paid off tremendously, providing the foundation for much of the lab’s current research. The process of glutamate receptor localization requires channel assembly and export from the ER, anterograde trafficking from cell body to synapse, anchoring at the synapse, endocytosis, recycling, and finally degradation. We obtained mutants for genes that regulate all of these steps in GLR-1 localization. Using additional subcellular markers, we showed that nearly all of the genes identified by this screen are relatively specific for GLR-1 localization, and do not impair protein trafficking or synapse formation in general. We have mapped and cloned many of these genes during the last few years, and describe several of them below.

The Role Of Cytosolic Tail Sequences And Subunit Interactions.

Many of the factors that are thought to facilitate glutamate receptor localization do so by interacting with carboxy-terminal tail sequences of these receptors. However, this hypothesis had been difficult to test for AMPA-type glutamate receptors, which can form heterotetrameric complexes with other subunits, making it difficult to assay the contribution of a single subunit. Our work with GLR-1 and a related subunit, GLR-2, indicated that subunit interactions can mediate glutamate receptor localization, and can mask the loss or removal of any one subunit within a channel. A subunit expressed in the absence of other subunits can become localized; moreover, it requires its carboxy-terminal tail sequences for this localization. In the case of GLR-2, we showed that these sequences, when placed on a heterologous transmembrane protein, are sufficient to confer localization. We are currently characterizing proteins that bind to the GLR-1 and GLR-2 tail sequences.

A Specific Requirement For The Unfolded Protein Response (UPR).

Subunit assembly is thought to occur in the ER. To our surprise, we found that the UPR pathway is required for glutamate receptors (but not other membrane proteins) to exit the ER, even in the absence of stress-inducing events. Our results demonstrated a role for the UPR in the absence of its previously-described response to ER stress induction, and showed that it can regulate the ER export of specific subsets of transmembrane proteins, as apposed to functioning simply as a general quality control for all secretory proteins that move through the ER. This work is supported by similar findings in mammalian neurons by Dr. David Bredt. Interestingly, human mutations in UPR components have been implicated in two disorders of the nervous system: bipolar disorder and Alzheimer’s disease. Our results raised the possibility that part of the pathology of these diseases could be due to improper glutamate receptor trafficking.

Receptor Turnover.

The lab of Dr. Josh Kaplan showed that GLR-1 is ubiquitinated, leading to endocytosis and proteolysis. My lab has identified several genes in the ubiquitin-proteasome pathway that have a relatively specific role in GLR-1 turnover. Our current studies focus on the specific targets of the ubiquitin-proteasome pathway, and the consequence for the receptors after ubiquitination.

Localization Dynamics: New Tools For Looking At Receptor Trafficking.

The requirement for ubiquitin-mediated endocytosis of GLR-1 suggests the importance of receptor dynamics in building and regulating the synapse. To better understand this process, we have been using Fluorescence Recovery After Photobleaching (FRAP) to monitor the rate of GLR-1 synaptic delivery. We have also used photoactivatable GFP (PAGFP), a variant developed by Dr. Lippincott-Schwartz that fluoresces only after photoactivation, to tag GLR-1 and follow its turnover via pulse-chase analysis. Finally, we have adapted an antibody injection protocol to measure the amount of surface-exposed GLR-1. We have analyzed GLR-1 localization dynamics in live lin-10 mutants using these tools, and have found that LIN-10 functions by stimulating the recycling of endocytosed GLR-1 receptors.

The Role of MAGUK Proteins.

Model system studies work best in collaboration with groups studying similar questions in mammals. Thus, our lab has been collaborating with another Rutgers professor who studies protein trafficking and PDZ-domain proteins in rat hippocampus: Dr. Bonnie Firestein. Our collaboration affords Dr. Firestein the opportunity to study C. elegans orthologs of mammalian proteins using gene knockdown, and our group the chance to study mammalian orthologs of proteins identified in our screens in cultured hippocampal neurons. We have published our collaborative studies on C. elegans orthologs of cypin and SAP97, showing a new role at the adherens junction for these proteins. We have also helped Dr. Firestein’s group analyze PSD-95 clustering using approaches developed in our lab.

Future Studies.

Only by identifying all of the factors that regulate GluR localization can we begin to have a complete understanding of this process. Thus, our current work focuses on mapping and characterizing the remaining genes identified in our screens. So far we have identified molecules that function intrinsically to regulate GluR localization. We are now beginning to focus on characterizing molecules that function extrinsically (e.g., cell-cell signaling molecules), with an emphasis on understanding how intrinsic factors respond to extrinsic signaling to regulate GluR localization. Finally, we are beginning to apply our newly-developed tools for analyzing GluR dynamics to our collection of mutants. Understanding these dynamics will provide critical insight to synaptic regulation.