Next generation RNA sequencing of Aβ-treated primary cultured mouse astrocytes to study the effect of ODC1 inhibition on AD-like astrocytes
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ABSTRACT:
PROVIDER: PRJNA771360 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16339857_1.fastq.gz | Fastqsanger.gz | |||
| SRR16339857_2.fastq.gz | Fastqsanger.gz | |||
| SRR16339858_1.fastq.gz | Fastqsanger.gz | |||
| SRR16339858_2.fastq.gz | Fastqsanger.gz | |||
| SRR16339859_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Reactive astrogliosis is a well-known and recognised marker of Alzheimer's Disease (AD). The aim of this study is to functionally visualise reactive astrocyte-mediated neuronal hypometabolism in the brains that is associated with AD and neuroinflammation. To investigate alterations of acetate and glucose metabolism and the effect thereof in AD-like astrocytes, primary astrocyte cultures were treated with acetate in the presence or absence of amyloid beta (Aβ) oligomers. On comparison, we found that acetate treatment upregulated the expression of acetate transporter MCT1 (Slc16a1) as well as that of enzymes involved in the urea cycle and subsequent GABA synthesis. We could, therefore, conclude that MCT1-mediated uptake of acetate into astrocytes triggered GABA synthesis by upregulating enzymes involved in putrescine production and degradation.
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Project description:The major genetic risk factor for Alzheimer’s disease (AD), APOE4, accelerates beta-amyloid (Aβ) plaque formation, but whether this is caused by APOE expressed in microglia or astrocytes is debated. We express here the human APOE isoforms in astrocytes in an Apoe-deficient AD mouse model. This is not only sufficient to restore the amyloid plaque pathology but also induces the characteristic transcriptional pathological responses in Apoe-deficient microglia surrounding the plaques. We find that both APOE4 and the protective APOE2 from astrocytes increase fibrillar plaque deposition, but differentially affect soluble Aβ aggregates. Microglia and astrocytes show specific alterations in function of APOE genotype expressed in astrocytes. Our experiments indicate a central role of the astrocytes in APOE mediated amyloid plaque pathology and in the induction of associated microglia responses.
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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.
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Project description:Microgliosis and astrogliosis characteristically occur at Aβ plaques in the brains of Alzheimer’s disease (AD) patients although the impact of gliosis in AD is poorly understood. We studied the impact of Aβ-induced gliosis using human induced pluripotent stem cell (hiPSC)-derived 3D neurospheres (hiNS), containing only astrocytes and neurons versus hiNS with the addition of iPSC-derived microglia (hiMG). Applying Aβ to hiNS containing only astrocytes and neurons triggered pathological features of AD including plaque-like aggregates, reactive astrocytes, oxidative stress, neuronal dysfunction and cell death. In contrast, when hiMG were combined with hiNS, infiltrating hiMG effectively phagocytose Aβ and facilitate neuroprotection. Our findings further support a pivotal role for microglia in modulating astrocyte Aβ responses by inducing AD-associated gene expression in astrocytes, including the upregulation of APOE, crucial for Aβ clearance. This model, highlighting the neuroprotective potential of microglia-astrocyte interactions, offers a novel platform for exploring AD mechanisms and novel therapeutic strategies.
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Project description:Next-generation RNA sequencing of Acetate treated primary cultured mouse astrocytes in the presence or absence of Aβ
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Project description:Alzheimer's disease (AD)-associated allele, apolipoprotein E4 (APOE4), exhibits pathological characteristics in an age-dependent manner despite its presence from birth. The molecular and cellular changes leading to disease onset are largely unexplored. We observed using human-induced pluripotent stem cells (hiPSCs)-derived models that astrocytes acquire immune priming functions through repetitive inflammatory stimuli, with recovery periods, causing transcriptome changes similar to those in the aged brain. Astrocytic priming by amyloid-β (Aβ) promoted cytokine release, leading to enhanced microglial phagocytic activity against Aβ. Conversely, APOE4 astrocytes showed impaired priming and reduced cytokine secretion in response to Aβ, leading to decreased microglial Aβ phagocytosis. Further, using a humanized APOE knock-in mouse model, we validated the positive effect of astrocyte priming on Aβ clearance in vivo. Our findings suggest that aging astrocytes may enhance Aβ clearance through the acquisition of immune priming functions and positively regulate Aβ clearance, while APOE4 impairs this process, leading to Aβ accumulation.
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Project description:Alzheimer's disease (AD)-associated allele, apolipoprotein E4 (APOE4), exhibits pathological characteristics in an age-dependent manner despite its presence from birth. The molecular and cellular changes leading to disease onset are largely unexplored. We observed using human-induced pluripotent stem cells (hiPSCs)-derived models that astrocytes acquire immune priming functions through repetitive inflammatory stimuli, with recovery periods, causing transcriptome changes similar to those in the aged brain. Astrocytic priming by amyloid-β (Aβ) promoted cytokine release, leading to enhanced microglial phagocytic activity against Aβ. Conversely, APOE4 astrocytes showed impaired priming and reduced cytokine secretion in response to Aβ, leading to decreased microglial Aβ phagocytosis. Further, using a humanized APOE knock-in mouse model, we validated the positive effect of astrocyte priming on Aβ clearance in vivo. Our findings suggest that aging astrocytes may enhance Aβ clearance through the acquisition of immune priming functions and positively regulate Aβ clearance, while APOE4 impairs this process, leading to Aβ accumulation.
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Project description:Alzheimer's disease (AD)-associated allele, apolipoprotein E4 (APOE4), exhibits pathological characteristics in an age-dependent manner despite its presence from birth. The molecular and cellular changes leading to disease onset are largely unexplored. We observed using human-induced pluripotent stem cells (hiPSCs)-derived models that astrocytes acquire immune priming functions through repetitive inflammatory stimuli, with recovery periods, causing transcriptome changes similar to those in the aged brain. Astrocytic priming by amyloid-β (Aβ) promoted cytokine release, leading to enhanced microglial phagocytic activity against Aβ. Conversely, APOE4 astrocytes showed impaired priming and reduced cytokine secretion in response to Aβ, leading to decreased microglial Aβ phagocytosis. Further, using a humanized APOE knock-in mouse model, we validated the positive effect of astrocyte priming on Aβ clearance in vivo. Our findings suggest that aging astrocytes may enhance Aβ clearance through the acquisition of immune priming functions and positively regulate Aβ clearance, while APOE4 impairs this process, leading to Aβ accumulation.
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