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Single cell RNA sequencing analysis of human Alzheimer’s disease brain samples reveal neuronal and glial specific cells differential expression

ABSTRACT: Alzheimer’s disease (AD) is the most common dementia worldwide with no current treatment and poorly understood molecular mechanisms. RNA expression studies suggest that AD is determined by diverse molecular events. Single cell technology allows comprehensive investigation of gene expression and cell specific changes in high precision of large number of brain cells. Computational analysis can reveal affected specific cell type marker genes and generate classification of the samples and gene networks. Here, we extensively characterized altered gene expression changes in post mortem brain samples of 2 individuals with AD (the brain samples originating from 3 brain regions, BA41/42,BA 6/8 and anterior hippocampal cortex). Wed detected significant changes in astrocyte marker genes. The samples were taken from patients in Braak stage III-IV. We found significant gene expression changes between the AD compared to the 2 control sequenced samples. We found significant changes in oligodendrocyte, microglia and astrocyte marker genes and several specific genes that were altered including the neuronal differentiation factor SOX2. Overall, our study thus reinforces a growing body of evidence implicating glia in AD, and highlights the need for greater mechanistic understanding of neuron-glia interactions in AD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE175814 | GEO | 2023/03/08

REPOSITORIES: GEO

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Project description:Unravel the mechanisms underlying brain aging and Alzheimer´s disease (AD) has been difficult because of complexity of the networks that drive these aging-related changes. Analysis of the gene expression in the brain is a valuable tool to study the function of the brain under normal and pathological conditions. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important research tool that potentially can provide the investigative power needed to address the complexity of brain aging and neurodegenerative processes. One of the reasons that account for the resistance of AD pathogenesis to analysis is that clinically normal subjects may exhibit considerable AD pathology, blurring criteria for distinguishing subjects with normal aging or AD. Here, we analyzed hippocampal and cortex frontal gene expression from 32 subjects separated in individuals presenting, 1) both pathologic and clinical AD (definitive AD); 2) AD pathology and normal clinic (pathologic AD); 3) cognitive impairment, without AD pathology (others dementias); and 4) no cognitive impairment, without AD pathology (normal individuals). Our results show that based on gene expression profile these individuals we could verify similarity between the definitive AD group and the group that only had AD-type pathology (pathologic AD). Specimens of hippocampus and cortex frontal used in this study were obtained at autopsy from 32 subjects. Neuropathology, specifically NFT and NP, was assessed in accordance to the Braak staging and CERAD scores, respectively. Based on pathologic and clinic criteria, subjects were categorized into four groups: 1) nine subjects with AD neuropathologic (Braak = IV / V / VI and CERAD ≠ 0) presenting cognitive impairment (CDR ≥ 1), termed “definitive AD” (dAD); 2) five subjects with AD neuropathologic (Braak = IV / V / VI and CERAD ≠ 0) and without cognitive impairment (CDR = 0), termed “pathologic AD” (pAD); 3) nine subjects without AD neuropathologic (Braak = 0 / I / II and CERAD ≠ C) presenting cognitive impairment (CDR ≥ 1), termed “other dementias” (OD); 4) - nine subjects without AD neuropathologic (Braak = 0 / I / II and CERAD ≠ C) and without cognitive impairment (CDR = 0), termed “normal” (N). RNA isolation and Amplification. From total RNA, a two-round linear amplification procedure (T7-based protocol) was carried out for all samples and for a pool of RNAs obtained from 15 distinct human cell lines used as reference. Labeled cDNA was generated in a reverse transcriptase reaction using amplified RNA (aRNA). Equal amounts of test and reference cDNA reverse color Cy-labeled aRNA targets were competitively hybridized against the cDNA probes in a customized cDNA platform with 4,608 ORESTES representing human genes. Dye-swap was performed for each sample as control for dye bias and used as replicate.

Project description:Alzheimer’s disease (AD), the most common age-related neurodegenerative disease, is closely associated with and manifested by neuroinflammation, yet the alteration of immune landscape in AD is largely unknown, preventing a deeper mechanistic understanding of neuroinflammation in AD. Two thirds of AD patients are females, and women have a higher risk of developing AD. Women with AD have more extensive brain histological changes than men with AD, more severe cognitive symptoms, and more severe neurodegeneration, suggesting that the disease affects female and male brains differentially. Despite evident AD sex variance, mechanisms and pathways are still poorly understood. Thus, a focus on sex differences in AD is essential to move the field towards effective interventions and to develop sex-specific therapies. To understand the cellular heterogeneity and disease-associated cellular changes in human AD brain, we performed unbiased massively parallel single nucleus RNA sequencing with postmortem frozen human brain tissues of middle temporal gyrus, a brain cortical region strongly affected by AD. From 12 individuals with and without AD, we isolated brain nuclei by sucrose gradient ultracentrifugation, generated single nucleus libraries with 10x Chromium platform (10x Genomics), and sequenced on NovaSeq6000 S4 sequencer (Illumina). We integrated snRNA-seq data of human brains from these 12 individuals of both Alzheimer’s Disease (AD, Braak Stage V/VI, n = 6) and age-matched healthy controls (HC, Braak Stage I/II, n = 6) by single nucleus analysis using Seurat. After quality control filtering, we profiled and analyzed 64,845 single nucleus transcriptomes, clustered all the cells jointly across the 12 donors that include 6 females and 6 males, and identified and annotated the major cell types of the human brain by interrogating the expression patterns of known gene markers, including neurons, excitatory neurons, inhibitory neurons, astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells, endothelial cells, and pericytes.

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Single cell RNA sequencing analysis of human Alzheimer’s disease brain samples reveal neuronal and glial specific cells differential expression

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