published_or_final_version / Physiology / Master / Master of Medical Sciences

Glutamate is the most abundant excitatory neurotransmitter in the mammalian nervous system. Ionotropic glutamate receptors used to be the only type of glutamate receptors, bringing about essential functions including synaptic transmissions. Since 1991, eight metabotropic glutamate receptors have been discovered. Belonging to the subfamily C of G protein-coupled receptor (GPCR) superfamily, these receptors have unique structural features. They couple to their own specific G proteins and transduce signals via pathways not recognized in other subfamilies. To date, little information on these receptors have been revealed in mammals, and even less is known about them in non-mammalian species including chicken. In the present study, various cDNAs of the chicken glutamate receptor, metabotropic 1 (GRM1) as well as its splice variants were cloned from adult brain tissue. At least 11 exons were identified in the chicken (c-) GRM1 gene, in which the alternative usage of exons and splice acceptor sites results in at least three variants, namely cGRM1a, cGRM1b and cGRM1f. The predicted coding regions of cGRM1a, cGRM1b and cGRM1f are 3459 base pairs (bp), 2736 bp and 2697 bp in length, which were deduced to encode receptor peptides of 1152 amino acids (aa), 911 aa and 898 aa, respectively. The predicted cGRM1a peptide shows high amino acid sequence identities (87.5% to 88%) to its counterparts in humans, rats, mice, chimpanzees and cattle. cGRM1b transcript differs from cGRM1a transcript by inclusion of two additional exons (7b and 7c), which contains a premature stop codon and results in its shorter C-terminal tail. cGRM1f is a novel splice variant that lacks exon 7b and is 13 aa shorter than cGRM1b. Reverse transcription-polymerase chain reaction (RT-PCR) assays showed that the transcripts of cGRM1a, cGRM1b and cGRM1f were preferentially expressed in adult chicken brains, in which cGRM1f mRNA was additionally identified in pituitary, lungs and gonads. Functional assay demonstrated that cGRM1a and cGRM1b receptors, expressed in Chinese hamster ovary cells, were induced by glutamate in dose-dependent manners via the Fura-2 dye calcium assays. In addition, dual luciferase reporter assays suggested that cGRM1a and cGRM1b receptors have no significant effects on the activation of cAMP/PKA and MAPK/ERK signaling pathways upon glutamate treatment. Taken together, the present study has provided the first step in understanding the possible roles of GRM1 in chickens. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Abstract not available

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

Fast excitatory neurotransmission is critical for learning and memory, and its dysregulation is linked to numerous neurological diseases. These include developmental diseases such as fragile X syndrome, psychiatric disorders like schizophrenia, and chronic neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Throughout the central nervous system, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors (AMPARs) mediate the fastest excitatory neurotransmission. In response to the neurotransmitter glutamate, AMPARs open their ion channels and allow cation flux through the post-synaptic membrane. This initiates rapid depolarization and signaling in the post-synaptic neuron. Nearly all AMPARs exist as complexes with auxiliary subunits, which are regulatory proteins that modulate receptor assembly, trafficking, pharmacology and function. These auxiliary subunits determine brain region-specific AMPAR signaling, and aberrancies in complex formation or function lead to neuropathologies. Despite their importance for CNS signaling and implication in neurologic disorders, the structural bases underlying the function of AMPARs and AMPAR complexes remain ambiguous, representing a critical barrier to our understanding of excitatory neurotransmission. As a consequence, structure-based design of neuro-therapeutics is largely undeveloped: there is only a single FDA-approved drug targeting AMPARs. To address these problems, I wanted to dedicate my thesis work to study AMPAR synaptic complexes across an array of functional states and provide a new foundation for our structural understanding of AMPAR signaling. First, I designed a covalent-fusion construct approach to guarantee assembly and expression of AMPAR synaptic complexes in heterologous cells (HEK293). Then, I developed purification protocols allowing me to obtain chemically homogenous and pure complex protein. Since synaptic signaling is highly dynamic, complexes of AMPARs with auxiliary subunits are conformationally heterogeneous and are not amenable to X-ray crystallography. Cryo-electron microscopy (cryo-EM) enabled me to approach these complexes structurally, where I could collect data and parse out heterogeneity through image classification. With cryo-EM, I solved the structure of an AMPAR bound to the auxiliary subunit stargazin, which promotes AMPAR activation. This work provided the first structural information on how AMPARs form complexes with regulatory subunits. In a following study, I solved the structure of an AMPAR in complex with a functionally distinct auxiliary subunit, GSG1L. In contrast to stargazin, GSG1L promotes inactivation and desensitization of AMPARs, thus having a neuroprotective effect. To further characterize the function of these auxiliary subunits, I designed chimeras between stargazin and GSG1L and examined their function electrophysiologically. This experiment revealed that AMPAR auxiliary subunits have a modular design, where variable extracellular domain regions, supported by a conserved transmembrane α-helical bundle, distinctly regulate function of the core AMPAR. This study provided the first evidence of how brain region-specific expression patterns of similarly-structured auxiliary subunits may contribute to unique AMPAR functions. More recently, I’ve taken advantage of the modulatory effects of stargazin on AMPARs and I applied cryo-EM to an AMPAR-stargazin complex. This study determined how AMPARs are activated by the neurotransmitter glutamate, and revealed a novel mechanism by which glutamate binding induces opening of AMPAR ion channels. Our data show that two-fold symmetric kinking of ion channel helices allows cation flux into neurons, which triggers neurotransmission. Importantly, this study also provides insights into how mRNA editing and patient-derived disease mutations in the transmembrane (i.e., resulting in aberrantly firing of receptors during epilepsy) reshape AMPAR function and excitatory neurotransmission. Collectively, the findings from my thesis work provide a new paradigm for the molecular-level understanding of glutamatergic neurotransmission throughout the CNS. These studies lay the groundwork for new directions in precision-medicine design of therapeutics targeting brain region-specific AMPAR synaptic complexes in neurological diseases.