2012 |
Parker, Whitney Erin |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
The Role of Stradalpha in Neuronal Migration and Brain Development @ University of Pennsylvania
DESCRIPTION (provided by applicant): Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is a severe neurodevelopmental disorder characterized by autism, intractable epilepsy, and a high rate of childhood mortality, recently identified in the Old Order Mennonite community of Lancaster County, PA. PMSE results from a homozygous deletion of the STRADa gene, encoding the protein STRADa, which serves as an upstream regulator of the mammalian target of rapamycin (mTOR) pathway. Loss of mTOR inhibition is correlated with several more common neurodevelopmental disorders, including Tuberous Sclerosis Complex (TSC) and Focal Cortical Dysplasia (FCD). Immunohistochemical analysis of PMSE post-mortem brain tissue reveals evidence of mTOR hyperactivity and failed neuronal migration, suggesting a possible common pathogenic mechanism. Therefore, STRADa was introduced as a key mTOR regulator critical for normal brain development. However, the mechanism by which it mediates this process is not understood. This proposal aims to define the role of STRADa in neuronal migration and cortical development, and thereby identify pharmacological targets for mTOR-associated neurodevelopmental disorders. Only a single PMSE post-mortem specimen has been available for analysis, and no STRADa knockout (KO) models currently exist. As a consequence, the cause of PMSE-associated neuropathology remains yet unknown. The proposed research will generate a novel STRADa KO mouse strain as a key tool for defining STRADa's function. The focus of Aim 1 is to characterize brain development in the STRADa KO mouse and identify the extent to which aberrant neuronal migration is dependent upon mTOR signaling. The focus of Aim 2 is to define in vitro the mechanism by which STRADa mediates neuronal migration using a novel neural progenitor cell migration assay. Cumulatively, the proposed experiments will identify STRAD¿ as a critical regulator of mammalian cortical development. PUBLIC HEALTH RELEVANCE: Many patients with intractable epilepsy and autism suffer from neurological disorders associated with hyperactive mTOR signaling during development. Investigating STRAD¿, as a critical regulator of mTOR, will reveal not only the mechanism by which this protein and signaling pathway control normal brain development, but also novel therapeutic targets for treating patients with otherwise intractable symptoms.
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1 |
2018 |
Parker, Whitney Erin |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
The Role of Novel Nmda Receptor Variants in Human Brain Development and Epilepsy @ Weill Medical Coll of Cornell Univ
NMDA receptor subunit NR2B plays a critical role in neurodevelopment, and has been found in animal models to influence synaptic pruning. Depletion of NR2B in radial glial neural progenitor cells (NPCs) in a rat model causes disrupted cortical lamination, though the mechanism is unknown. Patients with NR2B mutations com- monly manifest severe epilepsy and often cortical malformations suggestive of a neural migratory defect. How- ever, human models of NR2B function are entirely lacking, and treatments in vitro in in vivo in animal models have failed to translate into effective patient therapies. Two patients presenting to our Center for Neurogenetics with epileptic activity were found to carry distinct novel NR2B missense mutations, variants of uncertain signifi- cance (VUS). Fibroblasts from these patients will be used to model the effect of each VUS and create the first human model of NR2B mutation, to define the role of this protein in corticogenesis and synaptogenesis. It re- mains unknown whether and how each VUS contributes to the phenotype. Thus, in vitro analysis will be per- formed to determine the biochemical nature of each NR2B variant. Site-directed mutagenesis in NR2B cDNA plasmids and transfection into HEK-293 cells will be coupled with a cycloheximide pulse experiment to reveal each variant?s stability. Patient and wildtype (WT) fibroblasts will be reprogrammed into induced pluripotent stem cells (iPSCs), then differentiated into NPCs and glutamatergic neurons. Co-immunoprecipitation in NPC lines will determine the ability of each variant to bind NR1, requisite for the assembly of functional NMDA receptors. Mass spectroscopy will evaluate site-specific phosphorylation changes. CRISPR/Cas9 gene editing technology will be used to dissociate the mutation effects from background, by introducing each mutation into WT iPSCs, and by editing each mutation in patient lines via non-homologous end joining (NHEJ) and homology-directed repair (HDR). Neuronal cultures and cortical forebrain organoids will be derived from iPSCs to evaluate the functional effects of each mutant. Immunocytochemistry (ICC) will reveal the number of synapses per neurite, and the synaptic receptor composition. Patch-clamp recording will measure excitatory post-synaptic current (EPSC) amplitudes and action potential thresholds in each condition, and response to pharmacologic therapy, in a patient-specific model of epilepsy. NR2B mutant-derived cortical organoids will be assessed for disruption of neuronal migration in corticogenesis via immunohistochemical (IHC) staining for cortical layer markers, and cultured NPCs will be used to define the mechanism of NR2B?s role, through probing Rho GTPase activity, actin organization, and cell migration. Finally, calcium imaging will be performed on organoid slices, to evaluate net- work-level effects of NR2B mutations. These experiments will determine the impact of each novel NR2B muta- tion, to be contributed to the ClinVar database, define the mechanism of NR2B?s role in corticogenesis, establish the first human model of NR2B in synaptic development, and importantly, establish a platform for generating patient-based models of neurodevelopmental disorders for biological investigation and therapeutic assessment.
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0.921 |