Single-cell multiregion dissection of Alzheimer's disease

Single-cell multiregion dissection of Alzheimer's disease

Alzheimer's disease is the leading cause of dementia worldwide, but the cellular pathways that underlie its pathological progression across brain regions remain poorly understood. Here we report a single-cell transcriptomic atlas of six different brain regions in the aged human brain, covering one point three million cells from two hundred eighty-three post-mortem human brain samples across forty-eight individuals with and without Alzheimer's disease. We identify seventy-six cell types, including region-specific subtypes of astrocytes and excitatory neurons and an inhibitory interneuron population unique to the thalamus and distinct from canonical inhibitory subclasses. We identify vulnerable populations of excitatory and inhibitory neurons that are depleted in specific brain regions in Alzheimer's disease, and provide evidence that the Reelin signalling pathway is involved in modulating the vulnerability of these neurons. We develop a scalable method for discovering gene modules, which we use to identify cell-type-specific and region-specific modules that are altered in Alzheimer's disease and to annotate transcriptomic differences associated with diverse pathological variables. We identify an astrocyte program that is associated with cognitive resilience to Alzheimer's disease pathology, tying choline metabolism and polyamine biosynthesis in astrocytes to preserved cognitive function late in life. Together, our study develops a regional atlas of the ageing human brain and provides insights into cellular vulnerability, response and resilience to Alzheimer's disease pathology.

Alzheimer's disease is characterized by pathological protein aggregation in a stereotyped pattern across multiple brain regions. Post-mortem diagnosis of Alzheimer's disease is staged by the severity and distribution of these pathological hallmarks: extracellular amyloid-beta deposits and intracellular neurofibrillary tangles in neurons. Tangles are first seen in the entorhinal cortex (Braak stages one-two), then the hippocampus and thalamus (Braak stages three-four) and finally the neocortex (Braak stages five-six), a sequence that is typically synchronous with cognitive decline from mild cognitive impairment to severe dementia. Understanding the cellular architecture of affected brain regions has important implications for early and region-specific therapeutic interventions and may shed light on the molecular mechanisms underlying the regional progression of pathology. Although some brain regions relevant to Alzheimer's disease have been studied individually at scale or jointly in samples from a few individuals, a comprehensive molecular characterization of region-specific differences in Alzheimer's disease is currently lacking and could capture differences in regional molecular architecture and region-specific neuronal and glial subtype alterations in Alzheimer's disease and in cognitive resilience to Alzheimer's disease pathology.

Here we present a transcriptomic atlas of the human brain spanning six distinct anatomical regions from persons with and without Alzheimer's dementia as a basis for understanding disease-associated differences. We profile the transcriptomes of over one point three million nuclei from the entorhinal cortex, hippocampus, anterior thalamus, angular gyrus, midtemporal cortex and prefrontal cortex from forty-eight individuals, twenty-six of whom have a pathologic diagnosis of Alzheimer's disease. We annotate region-specific neuronal and glial subtype diversity, present an online resource for navigating this atlas and provide mechanistic insights into cellular vulnerability, response and resilience to Alzheimer's disease.

A multiregion atlas of Alzheimer's disease

To characterize cellular diversity in the human brain, and the genes, pathways and cell types that underlie Alzheimer's disease progression across brain regions, we performed single-nucleus RNA-sequencing analysis of nuclei isolated from two hundred eighty-three post-mortem brain samples across six brain regions from forty-eight participants in the Religious Order Study or the Rush Memory and Aging Project. We selected forty-eight participants on the basis of pathologic diagnosis of Alzheimer's disease (stratified by NIA-Reagan score of twenty-six with Alzheimer's disease and twenty-two without Alzheimer's disease; labelled non-Alzheimer's disease) and on the basis of clinical diagnosis of Alzheimer's dementia (n equals sixteen) versus non-dementia (n equals thirty-two) . From these forty-eight individuals, we profiled six brain regions: the entorhinal cortex (two hundred twenty-one thousand four hundred ninety-three cells), which is affected in early Alzheimer's disease (stages one-two); the hippocampus (two hundred twenty-one thousand four hundred fifteen) and thalamus (two hundred seven thousand six hundred twenty-five), which are affected in mid-Alzheimer's disease (stages three-four); and the angular gyrus (two hundred twenty thousand four hundred nine), midtemporal cortex (two hundred twenty-seven thousand four hundred twelve) and prefrontal cortex (two hundred fifty-four thousand seven hundred twenty-one), which are affected in late Alzheimer's disease (stages five-six), for a total of one point three five million transcriptomes of independent nuclei after the sample by subclass level . i, RNAscope validation of FOXP2 and MEIS2 as markers of the unique thalamus subtype, with quantification performed using Student's t-tests and representative images. The blue puncta represent MEIS2 (top) or FOXP2 (bottom) transcripts and red puncta represent GAD2 transcripts. FOXP2: n equals nineteen (prefrontal cortex) and n equals twenty-two (thalamus) cells; MEIS2: n equals thirty-five (prefrontal cortex) and n equals twenty-six (thalamus) cells; each dot represents an individual cell, pooled from eight samples (four individuals; each had one prefrontal cortex and one thalamus sample). j, Glutamatergic versus GABAergic scores for all neuron subtypes. The dotted lines represent the ninety-five percent confidence interval around the linear fit. P values were calculated using two-sided F tests. Ast., astrocytes; exc., excitatory neurons; inh., inhibitory neurons; mic., microglia/immune cells; olig., oligodendrocytes; vasc., vascular/epithelial cells.

removing doublets, low-quality cells and highly sample-specific clusters. We annotated seventy-six high-resolution cell types in fourteen major cell type groups, including thirty-two excitatory neuron subtypes (four hundred thirty-six thousand fourteen nuclei, thirty-two point two percent of total) and twenty-three inhibitory subtypes (one hundred fifty-nine thousand eight hundred thirty-eight nuclei, eleven point eight percent of total). We characterized these cell types in terms of their transcriptome size and proliferative status, compared our atlas with previously published data across species and identified broad cell type identity programs using non-negative matrix factorization and transcriptional regulons using SCENIC.

To gain insights into the cellular architecture of the human brain, we investigated differences in the composition of major cell types between the six brain regions. The fraction of neurons increased significantly from the TH (fourteen point four percent neurons) to the three-layer allocortical HC (thirty-two point two percent), the entorhinal periallocortex (thirty-six point six percent) and the six-layered neocortical regions (AG, MT and PFC, fifty-eight point nine percent). Glia, including astrocytes, oligodendrocytes, oligodendrocyte precursor cells and microglia/immune cells, tended to be less abundant in neocortical samples, in agreement with previous studies in humans and mice. Differences in the composition of major cell types between regions were reproducibly observed across study participants, irrespective of the individual's disease status, suggesting that variability in the major cell type composition between regions is a fundamental characteristic of the human brain and is not affected by AD pathology.