Final Report Summary - SALMANDNMDA (Elucidating the role of SALMs in the regulation of synapses and NMDA receptors)
Glutamate is the principal excitatory neurotransmitter in the mammalian brain. Cloning studies in the early 1990’s identified two distinct types of glutamate receptors, the metabotropic and ionotropic receptors. The primary ionotropic glutamate receptor subtypes are the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and N-methyl-D-aspartate receptors (NMDARs), which are present—usually together—at the postsynaptic membrane of most excitatory synapses in the mammalian brain. Our research is focused primarily on NMDARs. Recent evidence suggests that abnormal regulation of NMDARs plays a fundamental role in the development of many neurological and psychiatric disorders, including Alzheimer's disease, Parkinson’s disease and epilepsy. Thus, understanding the fundamental mechanisms that regulate the trafficking and function of NMDARs in the mammalian brain is essential for developing novel approaches to treat patients with these disorders.
NMDARs have five major subunits, GluN1 and GluN2A through 2D. A large body of evidence suggests that a functional NMDAR is a heterotetramer containing two GluN1 subunits and two GluN2 subunits. NMDARs are present both in excitatory synapses and at the extrasynaptic surface, and both the number and type of receptors are regulated at multiple levels, including protein synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking via the Golgi apparatus (GA), internalisation, recycling and degradation. Although much is known about functional and pharmacological properties of NMDARs, the mechanisms controlling their assembly and early trafficking events before they reach the cell surface membranes are poorly understood.
In this project, we examined two specific aims concerning the early trafficking of NMDARs, as outlined in the proposal:
Objective 1. It is expected that the functional NMDARs are assembled in the ER and after passing the ER quality control machinery, they are trafficked to the synaptic or extrasynaptic membranes. Different regions within both GluN1 and GluN2 subunits were shown to regulate the ER retention and release of functional NMDARs from the ER. However, it is not clear at what specific step(s) in the early biogenesis of the functional NMDAR these regions are employed and whether there are any subunit-dependent differences, e.g. between GluN1/GluN2A-B and GluN1/GluN2C-D receptors in the early processing of NMDARs.
In our studies, we found that:
(i.) the M3 domains of both GluN1 and GluN2 subunits contain key amino acid residues that contribute to the regulation of the number of surface functional NMDARs using truncated and mutated NMDAR subunits expressed in heterologous cells. These key residues are critical neither for the interaction between the GluN1 and GluN2 subunits nor for the formation of the functional receptors, but rather they regulate the early trafficking of the receptors. We also found that the identified key amino acid residues within both GluN1 and GluN2 M3 domains contribute to the regulation of the surface expression of unassembled GluN1 and GluN2 subunits. Thus, our data identify the unique role of the membrane domains in the regulation of the number of surface NMDARs.
(ii.) a single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit, L830, that regulates the surface number of NMDARs using chimeric and mutated GluN1 subunits expressed in heterologous cells. Our experiments show that this residue is not critical for the interaction between GluN1 and GluN2 subunits or for the formation of functional receptors, but rather that it regulates the forward trafficking of the NMDARs. The surface expression of both GluN2A- and GluN2B-containing receptors is regulated by the L830 residue in a similar manner. We also found that the L830 residue is not involved in the trafficking of individually expressed GluN1 subunits. Our data reveal a critical role of the single amino acid residue within the GluN1 M4 domain in the surface delivery of functional NMDARs.
(iii.) the surface expression of GluN1/GluN2C receptors is reduced compared to GluN1/GluN2A and GluN1/GluN2B receptors by combining microscopy and electrophysiology recordings of heterologous cells and cultured CGCs that express recombinant GluN subunits. Furthermore, using a panel of truncated and otherwise mutated GluN2C subunits, we identified three distinct regions in the GluN2C subunit—specifically, within the N-terminus, M3 domain, and C-terminus—that regulate the surface expression of GluN2C-containing NMDARs. Interestingly, trafficking of GluN1/GluN2A receptors is also regulated by the N-terminal and M3 domain‒mediated mechanisms; however, the C-terminal‒mediated mechanism appears to be specific to GluN2C-containing receptors. We conclude that the GluN2C subunit uses several regulatory mechanisms to control the early processing of functional NMDARs.
Objective 2. Nearly every protein that enters the ER lumen becomes N-glycosylated, including NMDARs. However, the identity of the N-glycans that are present on NMDARs and their role during the ER processing and early trafficking of NMDARs have not yet been investigated, despite the availability of several highly sensitive methods for analysing N-glycans. In the research proposed here, we combined state-of-the-art methodologies from two different fields—neurobiology and glycobiology—to examine the role that N-glycans play in the early trafficking of NMDARs.
In our studies, we found that:
(i.) NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (N203 and N368) are N-glycosylated using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy. Although GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.
(ii.) native NMDARs and AMPARs in cerebellar granule cells are N-glycosylated; moreover, unlike GluN2B and GluA1, the GluN1 subunit is fully sensitive to endoglycosidase H. Next, we used a full panel of lectins to determine the glycan composition of NMDARs and AMPARs in both cerebellar lysates and cultured cerebellar granule cells. Our experiments revealed 21 lectins that can immunoprecipitate both NMDARs and AMPARs; moreover, two and three lectins were specific for NMDARs and AMPARs, respectively. Our electrophysiological analysis revealed that several of these lectins can alter the functional properties of native NMDARs and/or AMPARs. These data provide new insight into the roles that N-glycosylation plays in regulating the function of glutamate receptors in the central nervous system.
I am confident that this project is highly relevant to the neuroscience research community, as: i) there is a current paucity of information regarding the regulation of early processing of ionotropic glutamate receptors; ii) similar multi-disciplinary approaches can be applied to investigate other membrane-bound proteins that are expressed in the mammalian brain; and iii) understanding mechanisms that regulate NMDAR function provide important clues to understanding many brain disorders.
Due to the obtaining of the MC-IRG, I have obtained a permanent position in our Institute and established my own independent research group, as envisioned in my MC grant proposal. My group currently consists of an experienced electrophysiologist - postdoc, Dr. Martina Kaniakova, two Ph.D. students, Katarina Lichnerova and Kristyna Skrenkova, and one undergraduate student, Sarka Danacikova. We published four original research studies, submitted one research study and wrote two review articles, all in respected international research journals. Moreover, we obtained several research awards including Best Diploma Thesis Award 2013 from Department of Physiology, Faculty of Science, Charles University in Prague (K. Lichnerova) and Otto Wichterle Award 2011 from Academy of Sciences of the Czech Republic (M. Horak). Furthermore, as proposed in my MC grant proposal, we established successfull international collaborations with Dr. Ronald S. Petralia at the NIDCD/NIH (USA), Dr. Young Ho Suh at Neuroscience Research Institute, Seoul National University College of Medicine in Seoul (South Korea) and Dr. Nathalie Sans at the Neurocentre Magendie in Bordeaux (France). Also, we secured enough funding to continue with our studies in future years and became tightly integrated into scientific and management structures of the Institute. All four team members are women and this project undoubtely helped them to learn new techniques, improve their scientific CVs and thus enhance their market value within European scientific community.
NMDARs have five major subunits, GluN1 and GluN2A through 2D. A large body of evidence suggests that a functional NMDAR is a heterotetramer containing two GluN1 subunits and two GluN2 subunits. NMDARs are present both in excitatory synapses and at the extrasynaptic surface, and both the number and type of receptors are regulated at multiple levels, including protein synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking via the Golgi apparatus (GA), internalisation, recycling and degradation. Although much is known about functional and pharmacological properties of NMDARs, the mechanisms controlling their assembly and early trafficking events before they reach the cell surface membranes are poorly understood.
In this project, we examined two specific aims concerning the early trafficking of NMDARs, as outlined in the proposal:
Objective 1. It is expected that the functional NMDARs are assembled in the ER and after passing the ER quality control machinery, they are trafficked to the synaptic or extrasynaptic membranes. Different regions within both GluN1 and GluN2 subunits were shown to regulate the ER retention and release of functional NMDARs from the ER. However, it is not clear at what specific step(s) in the early biogenesis of the functional NMDAR these regions are employed and whether there are any subunit-dependent differences, e.g. between GluN1/GluN2A-B and GluN1/GluN2C-D receptors in the early processing of NMDARs.
In our studies, we found that:
(i.) the M3 domains of both GluN1 and GluN2 subunits contain key amino acid residues that contribute to the regulation of the number of surface functional NMDARs using truncated and mutated NMDAR subunits expressed in heterologous cells. These key residues are critical neither for the interaction between the GluN1 and GluN2 subunits nor for the formation of the functional receptors, but rather they regulate the early trafficking of the receptors. We also found that the identified key amino acid residues within both GluN1 and GluN2 M3 domains contribute to the regulation of the surface expression of unassembled GluN1 and GluN2 subunits. Thus, our data identify the unique role of the membrane domains in the regulation of the number of surface NMDARs.
(ii.) a single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit, L830, that regulates the surface number of NMDARs using chimeric and mutated GluN1 subunits expressed in heterologous cells. Our experiments show that this residue is not critical for the interaction between GluN1 and GluN2 subunits or for the formation of functional receptors, but rather that it regulates the forward trafficking of the NMDARs. The surface expression of both GluN2A- and GluN2B-containing receptors is regulated by the L830 residue in a similar manner. We also found that the L830 residue is not involved in the trafficking of individually expressed GluN1 subunits. Our data reveal a critical role of the single amino acid residue within the GluN1 M4 domain in the surface delivery of functional NMDARs.
(iii.) the surface expression of GluN1/GluN2C receptors is reduced compared to GluN1/GluN2A and GluN1/GluN2B receptors by combining microscopy and electrophysiology recordings of heterologous cells and cultured CGCs that express recombinant GluN subunits. Furthermore, using a panel of truncated and otherwise mutated GluN2C subunits, we identified three distinct regions in the GluN2C subunit—specifically, within the N-terminus, M3 domain, and C-terminus—that regulate the surface expression of GluN2C-containing NMDARs. Interestingly, trafficking of GluN1/GluN2A receptors is also regulated by the N-terminal and M3 domain‒mediated mechanisms; however, the C-terminal‒mediated mechanism appears to be specific to GluN2C-containing receptors. We conclude that the GluN2C subunit uses several regulatory mechanisms to control the early processing of functional NMDARs.
Objective 2. Nearly every protein that enters the ER lumen becomes N-glycosylated, including NMDARs. However, the identity of the N-glycans that are present on NMDARs and their role during the ER processing and early trafficking of NMDARs have not yet been investigated, despite the availability of several highly sensitive methods for analysing N-glycans. In the research proposed here, we combined state-of-the-art methodologies from two different fields—neurobiology and glycobiology—to examine the role that N-glycans play in the early trafficking of NMDARs.
In our studies, we found that:
(i.) NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (N203 and N368) are N-glycosylated using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy. Although GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.
(ii.) native NMDARs and AMPARs in cerebellar granule cells are N-glycosylated; moreover, unlike GluN2B and GluA1, the GluN1 subunit is fully sensitive to endoglycosidase H. Next, we used a full panel of lectins to determine the glycan composition of NMDARs and AMPARs in both cerebellar lysates and cultured cerebellar granule cells. Our experiments revealed 21 lectins that can immunoprecipitate both NMDARs and AMPARs; moreover, two and three lectins were specific for NMDARs and AMPARs, respectively. Our electrophysiological analysis revealed that several of these lectins can alter the functional properties of native NMDARs and/or AMPARs. These data provide new insight into the roles that N-glycosylation plays in regulating the function of glutamate receptors in the central nervous system.
I am confident that this project is highly relevant to the neuroscience research community, as: i) there is a current paucity of information regarding the regulation of early processing of ionotropic glutamate receptors; ii) similar multi-disciplinary approaches can be applied to investigate other membrane-bound proteins that are expressed in the mammalian brain; and iii) understanding mechanisms that regulate NMDAR function provide important clues to understanding many brain disorders.
Due to the obtaining of the MC-IRG, I have obtained a permanent position in our Institute and established my own independent research group, as envisioned in my MC grant proposal. My group currently consists of an experienced electrophysiologist - postdoc, Dr. Martina Kaniakova, two Ph.D. students, Katarina Lichnerova and Kristyna Skrenkova, and one undergraduate student, Sarka Danacikova. We published four original research studies, submitted one research study and wrote two review articles, all in respected international research journals. Moreover, we obtained several research awards including Best Diploma Thesis Award 2013 from Department of Physiology, Faculty of Science, Charles University in Prague (K. Lichnerova) and Otto Wichterle Award 2011 from Academy of Sciences of the Czech Republic (M. Horak). Furthermore, as proposed in my MC grant proposal, we established successfull international collaborations with Dr. Ronald S. Petralia at the NIDCD/NIH (USA), Dr. Young Ho Suh at Neuroscience Research Institute, Seoul National University College of Medicine in Seoul (South Korea) and Dr. Nathalie Sans at the Neurocentre Magendie in Bordeaux (France). Also, we secured enough funding to continue with our studies in future years and became tightly integrated into scientific and management structures of the Institute. All four team members are women and this project undoubtely helped them to learn new techniques, improve their scientific CVs and thus enhance their market value within European scientific community.