Characterizing PTGER4 As a Target Gene of Autism Protein E6AP

Background: 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.

Objectives: This 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.

Methods: Potential 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.

Results: 1) 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.

Conclusions: We 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.