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Knockdown of microglial iron import gene, DMT1, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer's disease

ABSTRACT: Knockdown of microglial iron import gene, DMT1, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer's disease

PROVIDER: PRJNA1121187 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29317110_1.fastq.gz	Fastqsanger.gz
	SRR29317110_2.fastq.gz	Fastqsanger.gz
	SRR29317111_1.fastq.gz	Fastqsanger.gz
	SRR29317111_2.fastq.gz	Fastqsanger.gz
	SRR29317112_1.fastq.gz	Fastqsanger.gz

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Project description:Background Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer’s disease (AD). In vitro, microglia preferentially upregulate iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of knocking down microglial iron import gene, Slc11a2, on AD-related cognitive decline and microglial transcriptional phenotype. Methods In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-β-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1Cre-ERT2;Slc11a2flfl;APP/PS1+ or – mice were generated and administered corn oil or tamoxifen to induce knockdown at 5-6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12-15 months of age). Hippocampal CD11b+ microglia were magnetically isolated from female mice (15-17 months) and bulk RNA-sequencing analysis was conducted. Results DMT1 inhibition in vitro robustly decreased Aβ-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2KD APP/PS1 females displayed significant increases in genes Enpp2, Ttr, and iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2KD cells from APP/PS1 females also exhibited decreased expression of markers related to disease-associated microglia (DAMs), such as Apoe, Ctsb, Csf1, and Hif1α. Conclusions This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in female microglia and cognitive deficits in Slc11a2KD APP/PS1 mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.

Project description:Background: ABCA1 has recently been identified as a novel genetic risk factor for Alzheimer’s disease. The major known role of ABCA1 in the brain is to transfer lipids onto lipid poor APOE. Given that APOEε4 is the strongest genetic risk factor for developing late onset Alzheimer’s disease, this recent finding implicates the cholesterol homeostatic pathway in the onset/progression of Alzheimer’s disease. microRNAs (miRs) are small RNA species that negatively regulate expression of their target genes. ABCA1 is regulated by miR-33, and loss of this miR significantly increases protein levels of ABCA1 and increases the lipidation of APOE. However, it is unknown if loss of miR-33 and subsequent increase in ABCA1 protein levels ameliorates amyloid pathology. Methods: We generated miR-33+/+;APP/PS1 and miR-33-/-;APP/PS1 mice to determine changes in amyloid-beta (Aβ) peptides and amyloid plaque formation utilizing biochemical and histological analyses. In addition to amyloid pathology, we assessed if deletion of miR-33 altered the activation of glial cells in the brains of these mice. We utilized in vitro methods to determine if miR-33 alters the phagocytosis of Aβ aggregates. We utilized RNA-sequencing and mass spectrometry to identify the transcriptome and proteome regulation by miR-33 in the context of amyloid pathology. Results: Deletion of miR-33 dramatically reduces insoluble Aβ peptide levels and the deposition of amyloid plaques in APP/PS1 mice. In addition, we identified that astrocyte and microglial activation is markedly decreased in miR-33-/-;APP/PS1 mice through histological analyses. Intriguingly, we show that loss of miR-33 results in amyloid plaques that are more compact, potentially implicating enhanced microglial phagocytosis. We confirm in vitro that loss of miR-33 significantly increases microglial phagocytosis of Aβ aggregates. Our multi-omics analyses reveal that deletion of miR-33 regulates immune response in the context of amyloid pathology. Interestingly, we identified that WNT/Beta-catenin signaling is significantly upregulated in miR-33-/-;APP/PS1 mice. Conclusion: We confirm that the deletion of miR-33 increases APOE lipidation and significantly reduces amyloid pathology as well as glial activation in APP/PS1 mice. Our results agree with previous work identifying APOE lipidation as an important modulator of amyloid pathology. We show, for the first time, that loss of miR-33 increases the phagocytosis of Aβ by microglia. In total, we identify miR-33 as a promising target for the amelioration of amyloid pathology.

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Knockdown of microglial iron import gene, DMT1, worsens cognitive function and alters microglial transcriptional landscape in a sex-specific manner in the APP/PS1 model of Alzheimer's disease

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