Alu-Mediated Transcriptional Regulation in Neurodegeneration

Alu DNA sequences were from the GrCh37 (hg19) assembly. We designed unedited Alu DNA templates and changed A nucleotides edited in NCI HPC total vasculature but unedited in Alzheimer’s disease HPC total vasculature to G nucleotides as a mimic of A-to-I editing. Complete DNA sequences of unedited and edited MDM4 AluSg4 elements are shown in Supplementary Table 1. A SP6 promoter was added to the 5′ end and synthetic double-stranded DNA templates were obtained from IDT. RNA transcription was performed using Megascript SP6 (Invitrogen) essentially as previously described.^{46 (link)} THP-1 reporter cell lines (Invivogen) contain a stably integrated luciferase gene under the control of either an IFN-stimulated response element, ISRE or NF-κB response element. The human HMC3 microglial cell line was obtained from ATCC. THP-1 reporter or HMC3 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (HyClone), glutamine, and penn/strep at 37°C in a humidified atmosphere of 5% CO₂ in air. Transfections were performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific).^{28 (link)} Luciferase activity was determined after 24 h using luciferin substrate (Invivogen) and light emission measured with a TD20/20 luminometer. Gene expression measurements were performed essentially as previously described.^{25 (link),26 (link)}