Expression profiling by high throughput sequencing
Summary
Microglia are the tissue-resident macrophages of the central nervous system (CNS). Recent studies based on bulk and single-cell RNA sequencing in mice indicate high relevance of microglia with respect to risk genes and neuro-inflammation in Alzheimer’s disease. Here, we investigated microglia transcriptomes at bulk and single cell level in non-demented elderly and AD donors using acute human post-mortem cortical brain samples. We identified 7 human microglial subpopulations with heterogeneity in gene expression. Notably, gene expression profiles and subcluster composition of microglia did not differ between AD donors and non-demented elderly in bulk RNA sequencing nor in single-cell sequencing.
Overall design
To investigate transcriptomic changes in microglia during AD, bulk and single-cell RNA sequencing (scRNAseq) were performed. Post-mortem tissue samples of the superior parietal lobe (LPS) and superior frontal gyrus (GFS) were obtained from 27 donors. The samples were classified into 3 experimental groups based on a clinical diagnostic report provided by the Netherlands Brain Bank/ NeuroBiobank Born-Bunge and immunohistochemical analysis of Aβ and hyperphosphorylated tau (PHF-tau): CTR (no dementia, absence of Aβ plaques and PHF-tau), CTR with plaques (CTR+, no dementia, presence of Aβ plaques and/or PHF-tau) and AD (dementia, AD diagnosis, presence of Aβ plaques and/or PHF-tau). One donor diagnosed with mild cognitive impairment (MCI) and presence of Aβ and PHF-tau plaques was included in the single-cell studies. The stratification of CTR and CTR+ donors ensured that the CTR group was free of undiagnosed AD donors. Microglia were isolated from mechanically dissociated tissue using fluorescence-activated cell sorting (FACS) of single, viable CD11B and CD45 positive cells. Twenty-five microglia samples (13 LPS and 12 GFS) from 17 donors were sequenced as bulk samples, and 14 LPS samples from 14 donors were single-cell sequenced. Two donors were sequenced with both the 10X Genomics platform and the barcoded-SmartSeq2 protocol. Four donors (1 CTR, 3 CTR+) were included in both single-cell and bulk cohorts.
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