Animal experiments were conducted under the approval and guidance of the Institutional Animal Care and Use Committee (IACUC) at Washington University in St. Louis. The mice lived in a specific-pathogen-free (SPF) environment governed by the Division of Comparative Medicine at the Washington University School of Medicine, which is fully recognized by AAALAC International. Their living conditions were controlled, maintaining temperatures between 20-24 °C and a relative humidity of 40-60%. The mice were kept on a 12-hour light and dark cycle and had unrestricted access to standard lab chow and water. The Ube3a-deficient mice had been previously developed and were obtained from Jackson Laboratory. All the mice used in this research were of a C57BL6/J genetic background. Angelman mice (Ube3am−/p+) were produced by mating Ube3am+/p− heterozygous females with wild-type males. A heterozygous Asns mouse was created using guide RNA to delete a single nucleotide at the Q17 codon on exon 2 of the Asns gene. These mice were backcrossed with wild-type C57Bl/6J mice for a minimum of seven generations before their inclusion in this study. The injection of mRNA for Cas9 and the chosen gRNA was carried out into fertilized hybrid C57Bl/6J/JxCBA eggs, followed by confirmation through targeted Sanger sequencing. For genotyping, tail tips were incubated in a digest buffer and processed, leading to the purification of genomic DNA for subsequent PCR amplification and sequencing.
In terms of cell lines, HEK293T and Neuro2a cells obtained from ATCC were grown in a 5% CO2 humidified incubator and regularly screened for mycoplasma contamination. These cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with GlutaMAX, 5% fetal bovine serum, and antibiotic-antimycotic solution. Biochemical analyses were conducted after transfecting using the FuGENE 6 reagent. A stable UBE3A knockdown cell line was created in HEK293T using shRNA against the UBE3A gene, with control clones carrying a scrambled shRNA. While optimization aimed at improving knockdown efficiency, the stable clones showed a moderate reduction of about 25% in UBE3A expression, which, while modest, was adequate for inducing substantial biochemical changes in target proteins. Proteasome inhibition assays were performed using MG132 treatment on cells prior to analysis.
For primary cortical neuron cultures, the B-27 Plus neuronal culture system was employed. The process involved the isolation of embryos, dissociation of the cortex from both wild-type and Angelman mice, and seeding of cells on pre-coated culture dishes. The culture medium was refreshed every two days until experiments were conducted.
Various plasmids and AAVs were utilized, including MYC-tagged UBE3A isoforms cloned into a specific plasmid, driven by a chicken β-actin promoter, with verification via DNA sequencing. AAVs were created and packaged into AAV2/9n capsids and subsequently transduced into primary neurons.
In the realm of profiling and assays, HEK293T cells expressing certain UBE3A constructs were cultured with labeled amino acids for a comprehensive study. Following cell lysis and analysis steps involving mass spectrometry, differential expression analyses were conducted across patient-derived proteomic datasets. Western blot analyses and immunoprecipitations followed established protocols involving specific lysis buffers and primary antibodies.
Assays for in vitro ubiquitination were performed using recombinant proteins and involved various conditions for detection of ASNS ubiquitination. Immunofluorescence techniques were employed for examining the brain tissue of mice after fixation and sectioning, followed by staining with specific antibodies.
Behavioral assessments were performed on multiple mouse genotypes, observing various tests to gauge locomotion, anxiety, and other behavioral traits, conducted in a controlled environment. Statistical analysis was conducted across different measures to ensure data reliability, and RNA-seq analysis was executed to scrutinize gene expression at different developmental stages. The data from different methodologies were carefully summarized, reflecting a comprehensive approach to assessing their biological implications.
