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

ABSTRACT: 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 Aβ-treated astrocytes and further study the effect of DFMO treatment in Aβ-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 Aβ 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 Aβ treatment. Conclusions: Our study represents the first detailed analysis of mouse astrocyte culture transcriptome on Aβ-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 Aβ load in AD.

ORGANISM(S): Mus musculus

PROVIDER: GSE185916 | GEO | 2021/10/16

REPOSITORIES: GEO

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Project description:Astrocytes, one of the most resilient cells in the brain, transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in Alzheimer’s disease (AD). However, reactive astrocyte-mediated non-cell autonomous neuropathological mechanism is not fully understood yet. We aimed our study to find out whether Aβ-induced proteotoxic stress affects the expression of autophagy genes and the modulation of autophagic flux in astrocytes, and if yes, how Aβ-induced autophagy-associated genes are involved Aβ clearance in astrocytes of animal model of AD. Whole RNA sequencing (RNA-seq) was performed to detect gene expression patterns in Aβ-treated human astrocytes in a time-dependent manner. To verify the role of astrocytic autophagy in an AD mouse model, we developed AAVs expressing shRNAs for MAP1LC3B/LC3B (LC3B) and Sequestosome1 (SQSTM1) based on AAV-R-CREon vector, which is a Cre recombinase-dependent gene-silencing system. Also, the effect of astrocyte-specific overexpression of LC3B on the neuropathology in AD (APP/PS1) mice was determined. Neuropathological alterations of AD mice with astrocytic autophagy dysfunction were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through novel object recognition test (NOR) and novel object place recognition test (NOPR). Here, we show that astrocytes, unlike neurons, undergo plastic changes in autophagic processes to remove Aβ. Aβ transiently induces expression of LC3B gene and turns on a prolonged transcription of SQSTM1 gene. The Aβ-induced astrocytic autophagy accelerates urea cycle and putrescine degradation pathway. Pharmacological inhibition of autophagy exacerbates mitochondrial dysfunction and oxidative stress in astrocytes. Astrocyte-specific knockdown of LC3B and SQSTM1 significantly increases Aβ plaque formation and hypertrophic reactive astrocytes in APP/PS1 mice, along with a significant reduction of neuronal marker and cognitive function. In contrast, astrocyte-specific overexpression of LC3B reduced Ab aggregates in the brain of APP/PS1 mice An increase of LC3B and SQSTM1 protein is found in astrocytes of the hippocampus in AD patients. Taken together, our data indicates that Aβ-induced astrocytic autophagic plasticity is an important cellular event to modulate Aβ clearance and maintain cognitive function in AD mice.