Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment.
Nuclear receptors (NRs) are cellular proteins, which upon ligand activation, act to exert regulatory control over transcription and subsequent expression. Organized via systemic classification into seven subfamilies, NRs partake in modulating a vast expanse of physiological functions essential for maintenance of life. NRs display particular characteristics towards ubiquitination, the process of addition of specific ubiquitin tags at appropriate locations. Orchestrated through groups of enzymes harboring a diverse array of specialized structural components, the ubiquitination process emphatically alters the fate or downstream effects of NRs. Such influence is especially prominent in transcriptional processes such as promoter clearing for optimization and degradation pathways eliminating or recycling targeted proteins. Ultimately, the ubiquitination of NRs carries significant implications in terms of generating pathological clinical manifestations. Increasing evidence from studies involving patients and disease models suggests a role for ubiquitinated NRs in virtually every organ system. This supports the broad repertoire of roles that NRs play in the body, including modulatory conductors, facilitators, responders to external agents, and critical constituents for pharmacological or biological interventions. This review aims to cover relevant background and mechanisms of NRs and ubiquitination, with a focus towards elucidating subsequent pathophysiology and therapeutics in clinical disorders encompassing such ubiquitinated NRs.
BackgroundAutism spectrum disorder (ASD) has been found to be associated with duplication or triplication of the UBE3A gene, which encodes for E6‐associated protein (E6AP, an E3 ubiquitin ligase and transcriptional coactivator). Mice with three copies of UBE3A exhibit core ASD features and humans with Dup15q share many symptoms with ASD. However, the few E6AP ubiquitination substrates found do not explain ASD pathology. Our lab has identified and characterized E6AP as a coactivator of steroid hormone receptor signaling. Estrogens (E2) affect learning, memory and many other brain processes via estrogen receptors, which are transcription factors.ObjectivesThis study tests the hypothesis that deregulation of E6AP‐mediated steroid hormone receptor transcriptional signaling in the brain leads to the development of ASD. The project aims are to identify steroid hormone‐dependent E6AP target genes in neurons and to study the role of these target genes in the pathogenesis of ASD. Identification of new molecular pathways that are transcriptionally regulated by E6AP will broaden our understanding of ASD.MethodsPotential E6AP target genes were identified by microarray of MCF‐7 breast cancer cells. Cells from the mouse neuroblastoma Neuro2a cell line were cultured. Cells were transfected with E6AP or had E6AP knocked down by siRNA and then were treated with physiologically relevant doses of estrogen, the estrogen receptor antagonist tamoxifen, or vehicle. Assays included western blot, co‐immunoprecipitation, qRT‐PCR, and microscopy.Results1) E6‐AP and ER colocalize in mouse HPC neurons. 2) E6‐AP and ERα translocate to the nucleus of Neuro2a (N2a) cells upon E2 treatment. 3) E6‐AP and ERα complex in N2a. 4) The learning and memory gene for prostaglandin E receptor 4, PTGER4, is an E6AP‐dependent target gene that is downregulated in the presence of E6AP or estrogen.ConclusionsWe have identified a memory and learning gene that is regulated by E6AP and E2‐dependent: PTGER4. This is evidence that PTGER4 may be altered in ASD, leading to learning and memory symptoms. PTGER4 allows phosphorylation of glycogen synthase kinase 3 (GSK3). GSK3 has a large role in apoptosis and has been implicated in neuropsychiatric disorders such as Alzheimer's disease and bipolar disorder. Given that GSK3 is amyloidogenic and ASD patients exhibit increased beta amyloid deposition, increased E6AP leading to decreased PTGER4 may lead to decreased inhibitory phosphorylation of GSK3. In a case study, the GSK3 inhibitor ketamine actually improved an adult ASD patient's symptoms, supporting this theory. Further experiments will be necessary to confirm these promising findings.Support or Funding InformationUniversity of Miami
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