Next generation RNA sequencing of Aß-treated primary cultured mouse astrocytes to study the effect of ODC1 inhibition on AD-like astrocytes

Purpose: The existence and role of the urea cycle has not been well studied in the astrocytes of Alzheimer's Disease (AD). In the past, ODC1-inhibitor DFMO (Diflouromethylornithine) has been shown to have a neuroprotective effect on AD-like pathology, but the biochemical mechanism of this effect remains largely unknown. In this study, we aim to compare the transcriptome of naive and Aß-treated astrocytes and further study the effect of DFMO treatment in Aß-treatment condition. Methods: Primary astrocyte culture (from P0-P1 mouse pups) mRNA profiles were generated by high-throughput NGS in 3 treatment conditions in duplicate using HiSeq 2500. Sequence reads were analysed using Partek Genomics Suite, aligned using STAR and then quantified to the mouse genome assembly (mm10 Ensemble transcripts release 99), following which they were normalised to median ratio values and differentially analysed using the DeSeq2 algorithm. Finally, pathway analysis was carried out to study the differential expression of biomolecular pathways, with reference to the KEGG database. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the mouse genome (build mm10) and identified 18783 genes in the dataset. Differential analysis revealed upregulation of autophagy-related genes as well as switching-on of the genes involved in the cyclic metabolism of urea on Aß treatment of astrocytes. ODC1 inhibition by DFMO induced upregulation of genes involved in the non-amyloidogenic processing of APP and downregulation of amyloidogenic genes. Pathway analysis revealed upregulation of urea cycle and urea metabolic process pathways, as well as selective autophagy and phagosome-lysosome fusion on Aß treatment. Conclusions: Our study represents the first detailed analysis of mouse astrocyte culture transcriptome on Aß-treatment, and the effect of ODC1 inhibition on the same, with biological replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. From our results, we can conclude that the urea cycle is turned on in Alzheimer's Disease (AD)-like condition and that ODC1-inhibition can be a suitable therapeutic strategy to reduce the Aß load in AD. Overall design: RNA-seq of astrocytes untreated or treated with Aß or Aß with DFMO