Terunaga Nakagawa - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to develop and apply unique specimen preparation protocols and correlated imaging techniques to examine changes in activity-dependent dendritic spine morphology and synaptic ultrastructure.This work will propel development and application of the tetracysteine/biarsenical labeling system for correlated microscopy and of high resolution imaging, shaping development of methods used for processing, refinement and interpretation of information in reconstructions from electron tomography when specifically applied to the complex problem of deducing the molecular organization of synaptic structures.

DESCRIPTION (provided by applicant): The origin of life on our planet is widely believed to be the so-called "RNA world". During evolution, before DNA and proteins were part of life there was a world full of RNAs that possess self-replicating enzymatic ability. The history of RNA world is recorded in the current life. For example, ribosome is a peptide-bond forming enzyme whose catalytic core is formed exclusively by RNA. The proteins in the ribosomes have rather accessory and regulatory roles that are acquired later during evolution. The small RNA is another example that demonstrates the important regulatory function of RNA in various biological processes. How did lipid membrane join the RNA world? Cellular membranes have extremely important roles in providing the ideal conditions for the chemical reactions in the cytoplasm. However there is no convincing model that explains how membranes were integrated into life after the "RNA world". In this EUREKA proposal, I will test the hypothesis that some form of RNA exists that regulates the function of lipid bilayers. More specifically, I consider the existence of the following kind of RNAs. First, there may be a category of small RNA that regulates the function of plasma membrane. In another case, there may be primitive ion channels that are formed by RNA with accessory proteins. Protein conducting channels in the endoplasmic reticulum binds to ribosomes and therefore may be considered as one example of a system in which RNAs function at the membrane. Taken together there is a good chance that RNAs are embedded in the membrane and play fundamentally important function in biology. To test this hypothesis, we will investigate whether any RNA forms are co-purified from the brain membranes. The brain will be used as a model organ because it contains a rich variety of membranes. Two approaches will be taken;(1) We will biochemically enrich neuronal membranes and chemically strip off peripheral membrane attached proteins. We will detergent solubilize these membranes and isolate RNAs by separating them from transmembrane proteins. (2) The total RNA from brain will be reconstituted into membrane made of total brain lipids. The membranes will be separated from the unbound RNA by density gradient ultracentrifugation. The isolated membrane will be solubilized in detergent and further reconstituted into liposomes. By iteratively repeating lipid reconstitution, isolation, and solubilization, we will enrich membrane bound RNA. We will determine the sequence of the identified RNAs and search for the genomic database to verify that they are not protein coding RNAs nor ribosomal RNAs. If we will be successful in identifying such novel RNA forms that function in the membrane we will further pursue to define their precise functions in the membrane. The identification of RNAs in the membrane will add yet another entity of biological macromolecules that will revolutionize the way we describe biology and medicine. In particular, because brain has the highest lipid composition of all organs, we expect that the results of this research will strongly impact the understanding of the physiology and dysfunction of the nervous system. PUBLIC HEALTH RELEVANCE: This proposal aims to identify novel form of RNAs in the cellular membrane that possess fundamentally important biological functions such as those of ion channels, transporters, and structural regulators of membrane. A discovery of this kind of RNAs may explain novel phenomena mediated by RNA in the membranes in organs that are rich in lipids, such as brain. Because dysfunction of lipid metabolism and membrane morphology have been already implicated in various disorders, the results obtained form this project may deepen our understanding of a variety of diseases including, fragile-X mental retardation, schizophrenia, autism, and dementia.

DESCRIPTION (provided by applicant): Subunit assembly and vesicle trafficking of AMPA receptors (AMPA-Rs) play central roles in synaptic plasticity. The molecular basis for AMPA-R function, trafficking, and biogenesis remains, however, poorly understood. Current data in the field suggest that AMPA-Rs have a dimer-of-dimers organization, asymmetric overall structure, and in the brain contain auxiliary stargazin/TARP subunits that modulate AMPA-R function. To obtain more insight into AMPA-R structure and assembly, which are critical for understanding synaptic plasticity, we propose to compare the 3D EM structure of the mature GluR2 tetramer with those of dimeric biosynthetic intermediates. We will DOX dependently express GluR2 subunits in stable HEK cell lines, purify the recombinant receptors and study their structures by single-particle electron microscopy. In Aim 1 we will test whether normal subunit assembly requires the subunits to adopt a specific conformation and whether point mutations interfering with subunit assembly change the subunit conformation. First, we will work towards obtaining an improved 3D structure of a fully assembled AMPA-R by imaging tetramers formed by recombinant GluR2 flip and locked into the non-desensitized state. We will also determine the structure of immature GluR2 dimers. By comparing the 3D structures of the mature and immature AMPA-Rs, we will unveil the inter-domain contacts that drive the assembly of dimers into tetramers. Finally, we will study the 3D structures of dimer intermediates of mutant GluR2 subunits and compare those with the structures of wild-type GluR2, providing insight into how mutations may interfere with subunit assembly. Because in the brain auxiliary stargazin/TARP subunits are associated with AMPA-Rs, in Aim 2 we will investigate when stargazin binds to GluR2 during maturation and how stargazin modulates trafficking and subunit assembly of GluR2. We have established stable HEK cell lines that constitutively express stargazin but GluR2 only in the presence of DOX. We will compare the time course of the DOX-induced expression of GluR2 in the presence and absence of stargazin to quantitatively test if stargazin is a molecular chaperone of GluR2. We will further use GFP-tagged GluR2 in neurons to examine the effect of stargazin on receptor dynamics using time-lapse fluorescent confocal mircroscopy. Results from these experiments will determine the contribution of stargazin to the trafficking and maturation of newly assembled AMPA-Rs. The NMDA receptor (NMDA-R) is another member of the glutamate receptor family that plays critical roles in synaptic plasticity. Evidence in the field suggests that the domain arrangement and mechanism of subunit assembly may differ between NMDA-Rs and AMPA-Rs. In Aim 3, we will therefore use the approaches we established for our studies on AMPA-Rs to NMDA-Rs. In particular, we will use EM to study the structure of heterotetrameric NMDA-Rs formed by NR1 and NR2B subunits. PUBLIC HEALTH RELEVANCE: Dysfunction of glutamate receptors causes a variety of neurological and psychiatric disorders and stroke. Mutations in glutamate receptor subunits have been found in patients with X-linked mental retardation, and thus altered glutamate receptor function and assembly is likely to be the direct cause of the disease. By revealing the detailed molecular basis for wild-type and mutant glutamate receptor function, trafficking and subunit assembly, this proposal intends to extend our understanding of neurological and mental disorders, which may especially lead to curing X-linked mental retardation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to develop and apply unique specimen preparation protocols and correlated imaging techniques to examine changes in activity-dependent dendritic spine morphology and synaptic ultrastructure.This work will propel development and application of the tetracysteine/biarsenical labeling system for correlated microscopy and of high resolution imaging, shaping development of methods used for processing, refinement and interpretation of information in reconstructions from electron tomography when specifically applied to the complex problem of deducing the molecular organization of synaptic structures.

DESCRIPTION: Cognition, behavior, activity of neural circuits, synaptic plasticity, and neuronal survival relate to proper functioning of ligand gated ion channels of the ionotropic glutamate receptors (iGluRs). AMPA-type iGluRs (AMPA-Rs) contribute to the majority of excitatory synaptic transmission in mammalian brain and their dysfunction involves a variety of neurological and psychiatric disorders. For example, positive modulators of AMPA-Rs alleviate major depression disorder (MDD) symptoms, synaptic AMPA-Rs are reduced in mouse models of Alzheimer's disease, and auto-antibodies that bind to AMPA-Rs cause Rasmussen's encephalitis and a subset of limbic encephalitis. In particular, existing drugs for MDD targeting AMPA-Rs have limitations and mechanistically novel AMPA-R modulators are needed for therapeutic development and understanding disease mechanism. AMPA-Rs are protein complexes made of ? and ? subunits. The ? subunits are known as GluA1-4 and construct the tetrameric core of the ligand gated ion channel pore, whereas ? subunits contribute to functional modulation of the receptors without being part of the pore structure. The ? subunits are also known as the auxiliary factors and may be regarded as a set of endogenous modulators of AMPA-Rs developed by nature during evolution. Auxiliary factors modulate the magnitude and shape of postsynaptic responses mediated by AMPA-Rs. Mechanistically, modulating trafficking relates to changing the number and mobility of AMPA-Rs at synapses, whereas altering the gating kinetics of the ion channel will directly modify the time course of membrane depolarization. By changing trafficking and gating parameters influencing postsynaptic currents, auxiliary subunits impact coincident detection and dendritic integration. Ultimately such synaptic modulation is believed to affect the activity of neural circuits and behavior. It is thus conceivable that intervening with endogenous modulators of AMPA-Rs would have strong physiological effects. It is unclear, however, whether ? subunits of iGluR would be effective drug targets for manipulating ligand gated ion channel function. To test this hypothesis, we plan to combine HTS with new cell based assays we developed in order to screen for chemical compounds that specifically act on auxiliary factor dependent modulation of AMPA-Rs. Conventional drugs developed against AMPA-Rs focus on pore forming ? subunits. If new probes are identified from our proposed screening, auxiliary factors of iGluR will become a mechanistically new target for drug development. It is important to note that ? subunits in potassium channels are already established drug targets. New endogenous auxiliary factors of AMPA-Rs are continuously being identified, providing broader spectrum of molecular targets. Each auxiliary factor has distinct expression patters, indicating that targeted drugs will have cel-type specific effects. Using the proposed HTS screening approach, the identification of compounds that modulate AMPA-R activity through interaction with the auxiliary subunits will provide a paradigm shift in developing new drugs against AMPA-Rs, in particular for MDD.

The AMPA type ionotropic glutamate receptors (AMPARs), a ligand gated ion channel activated by the neuro- transmitter glutamate, mediate the majority of excitatory neurotransmission in the brain. The signals trans- duced by these complexes are critical for synaptic plasticity, learning and memory. AMPAR auxiliary subunits regulate trafficking and gating modulation of AMPARs. In this proposal we will investigate the mechanism of AMPAR regulation by their auxiliary subunits. The two major AMPAR auxiliary subunits, in the hippocampus, cortex, and striatum, are TARPs and cornichons (CNIHs). The TARPs are extensively studied and therapeutic compounds to alleviate seizure are already available to target ?-8 TARP, a hippocampus enriched TARP. On the other hand, our understanding on CNIHs is limited. Within the CNIH family, CNIH2/3 is known to function as AMPAR auxiliary subunits. In humans, the N-terminus of CNIH2 that forms the interaction interface with AMPAR is intolerant to missense mutations, indicating an essential role of CNIH2-AMPAR interaction in hu- mans. Our hypothesis is that CNIHs play fundamental roles in regulating AMPAR gating during synaptic transmission and plasticity. To further establish this hypothesis, we will study the functional mechanism of complexes made of GluA2 subunit of AMPAR and CNIH3 as a model. Our lab has recently solved the cryo-EM structure of GluA2/CNIH3 complex in GluA2:CNIH3=4:4 stoichiometry at high resolution. In Aim 1 we hypothe- size that the GluA2/CNIH3 complex could exists in other stoichiometry, and propose to reveal the architecture of complex in GluA2:CNIH3=4:2 stoichiometry using cryo-EM. CNIH1 is currently not categorized as AMPAR auxiliary subunit. However the cryo-EM structure of the GluA2/CNIH3 complex tells us that CNIH1 possess AMPAR binding motif that is present in CNIH2/3. The cryo-EM structure also revealed the presence of lipids surrounding the complex. We hypothesize that these lipids may play important functional roles in AMPAR gat- ing modulation. In Aim2 we will test roles of CNIH1 and lipids in gating modulation of AMPAR. Finally, we hy- pothesize that revealing the allosteric gating modulation mechanism of CNIH3 would require obtaining snap- shots of lipid embedded GluA2/CNIH3 complex in channel closed, open, and desensitized states. In Aim 3, we propose to solve high resolution cryo-EM structures of GluA2/CNIH3 complex embedded in a lipid bilayer mi- metic environment, and compare them in different functional states. The role of auxiliary subunits in tuning ion channel gating kinetics is predicted to have significant impact on circuit dynamics. In summary, the outcomes of this study are expected to advance our mechanistic understanding of AMPAR function and assist developing new therapeutic compounds that can alleviate dysregulation of AMPARs seen in neurological and psychiatric disorders, such as Alzheimer's disease, stroke, autism, Rasmussen's and limbic encephalitis, and seizure.

PROJECT SUMMARY We propose to acquire a Thermo Scientific Glacios, a 200kV cryo-transmission electron microscope, to advance our biological investigation using the growing technologies of cryo-EM. The Glacios will be a part of the Cryo-EM Facility, a shared resource of the Center for Structural Biology. Our investigators have established records of using single particle analysis and discovering novel biological molecular mechanisms at the atomic level. The addition of the Glacios will be essential for our investigators to further advance their research beyond what is currently possible using our existing instruments. Specifically, the Glacios will be essential for: (1) structural analyses of low molecular weight proteins utilizing 200kV acceleration voltage, (2) the MicroED approach that uses electron diffraction of micro-crystals, (3) investigation of macromolecular complexes in situ using cryo- electron tomography, and (4) specimen optimization to advance our efficiency to resolve at high- resolution fine conformational varieties and small molecules, such as ligands, drugs, and ions. The technology provided by the Glacios is essential for breaking new ground on the NIH-funded research projects of the major users, which include studies of synaptic transmission and plasticity (Nakagawa), in situ synapse architecture and synaptic vesicle fusion (Zhou), Clostridioides difficile toxin pathology (Lacy), nuclear mRNA transport (Ren), DNA replication and repair (Eichmann), calcium signaling (Karakas), in situ viral assembly and architectures (Wan), vesicle and membrane trafficking (Jackson), and bacterial signaling (Iverson). Additionally, 11 minor users plan to exploit the Glacios to investigate critical biological questions in membrane protein function, inflammatory signaling, genome maintenance, bacteria-host interactions, ion channel mediated signaling, lipid signaling and biosynthesis, and molecular motors. The outputs of the research projects that will use the Glacios will substantially advance our basic understanding on the mechanisms of human physiology, pathology, and disease therapeutics. The Glacios will initially be used by structural biologists and cell biologists, with the userbase predicted to extend into the fields of chemistry and bioengineering who have related scientific interests. In fact, the Glacios will be housed in the Engineering and Science Building, which is designed to nurture collaborations between disciplines via a three-floor, integrative, and collaborative laboratory space linking the School of Medicine, College of Arts and Sciences, and the School of Engineering.