2015 — 2021 |
Yuan, Hongjie |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Nmdar Mutations & Neurodevelopmental Disorder: From Mechanism to Targeted Therapy
DESCRIPTION (provided by applicant): Neurodevelopmental disorders are associated with disabilities in brain function that affect a child's behavior, memory or ability to learn. Such disabilities carry devastating mental, emotional, and economic consequences for the individuals, their families, as well as society. The molecular bases for a subset of disabilities involve disease-causing mutations in various ion channel families, including NMDA receptors (NMDARs). The cation-selective NMDAR channels formed from assembly of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits mediate a slow, Ca2+-permeable component of excitatory synaptic currents that can trigger changes in synaptic strength, a cellular correlate of learning. NMDARs also play an important role in normal brain development. A large number of mutations (>140) have been reported in just the last three years, leading to the view that these mutations are present in a subset of patients with neurological disorders, particularly early onset intractable seizures. Surprisingly, the incidence of NMDAR mutations found in pediatric patients presenting with neurological problems is 5.7%, similar to or higher than that for Na+, K+ , Ca2+ channels and GABA receptors. Mutations in NMDAR subunits have been identified in children with a broad range of neurodevelopmental problems, including attention deficit hyperactivity disorder (ADHD), autism spectrum disorders, developmental delay, mental retardation, schizophrenia, intellectual disability, and intractable seizures. Unfortunately, virtually no functional analysis of these mutations exists, making it impossible to evaluate effects of mutations in the context of clinical phenotype. We proposed 4 lines of experimentation addressing the molecular mechanism underlying neurological diseases suggested to arise from mutations in NMDAR subunits. We will study the functional effects of mutations in the transmembrane domain (TM), linkers, and ligand binding domains (LBD) and test the ability of FDA-approved drugs to rectify the mutation-induced gain-of-function. All experiments will utilize receptors that contain 0, 1, or 2 mutant NMDAR subunits, enabling an assessment of function in heterozygous patients. Aim 1. How do human NMDAR mutations in the TM- linker regions impact function? We will analyze 26 mutations in the transmembrane domain or associated linkers. We will collaborate on efforts to obtain crystals of the open channel configuration. Aim 2. How do human NMDAR mutations in the ligand binding domains impact function? We will evaluate the functional effects of 36 mutations in the ligand binding domain, and collaborate to obtain crystallographic data. Aim 3. How do human NMDAR mutations influence neuronal trafficking and function? We will analyze the properties of NMDAR-mediated synaptic current in slice cultures transfected with mutant NMDAR subunits. Aim 4. Are NMDAR channelopathies treatable? We will evaluate the potency (IC50) of FDA-approved NMDAR antagonists at gain-of-function NMDAR mutations and evaluate the neurotoxic potential of NMDAR mutations.
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2021 |
Yuan, Hongjie |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Human Gria Variants and Neurological Diseases: From Molecular Mechanism to Rescue Pharmacology
Modified SUMMARY-ABSTRACT Neuropsychiatric disorders are associated with disabilities of brain function that affect individual?s behavior, memory and ability to learn. Such disabilities can carry devastating mental and economic consequences for the individuals, their families, and society. The molecular basis of a subset of these disabilities involves monogenic channelopathies, a term used to describe disease-causing variants in various ion channels. The ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), ligand-gated ion channels, represent tetrameric complexes comprised of varying combinations of four subunits, GluA1-4 (encoded by GRIA1-4). AMPARs mediate the fast component of excitatory post-synaptic currents. Patterns of AMPAR activation can trigger a change in synaptic strength, which is widely considered to be a cellular correlate of learning and memory, and play an important role in neuronal development. Following the first report on a disease-causing AMPAR variant in 2007, a large number of human variants (>200) scattered across four AMPAR subunits have been identified in patients with various neurodevelopmental and neuropsychiatric problems, including autism and intellectual disability. It has been suggested that GRIA2 and GRIA3 genes have genome-wide significance for autism and schizophrenia, respectively. Despite the rapid advance in identification of new variants, there are neither virtually no systematic functional analyses for the variants nor any evaluation of possible treatment options for the patients. We propose a series of functional and pharmacological experiments that will fill this gap in our knowledge and will determine the mechanisms underlying the effects of 64 disease-associated GRIA2 and GRIA3 variants that do not exist in healthy population. The proposed experiments will explore how the receptor and neuronal function is impacted by genetic changes in AMPAR GRIA genes. The results of our pharmacological experiments assessing the effects of FDA-approved drugs on AMPARs with patient-specific variants will advance opportunities for personalized medicine by suggesting new therapeutic strategies for mitigation of functional changes by these variants. Our data will also provide novel functional insight into the AMPAR function. Aim 1. How do human GRIA variants impact receptor function? We assess the effect of 64 missense GRIA2 and GRIA3 variants on agonist potency, time course of current responses, and cell surface receptor trafficking. Aim 2. How do human GRIA variants influence neuronal function? We will assess neuronal synapse number, spine morphology, trafficking locations (synaptic vs extrasynaptic), spontaneous mEPSCs, and the ability of induced neurotoxicity (cell viability as well as dendritic swelling) by a set of GRIA2 and GRIA3 variants. Aim 3. How can AMPAR channelopathies best be treated? For the gain-of-function variants, we will measure the IC50 for competitive antagonists, negative allosteric modulators, or channel blockers (including FDA-approved). We will estimate the EC50 for positive modulators (e.g. ampakines) for the loss-of-function variants.
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