Abstract
A common approach to understanding neurodegenerative disease is comparing gene expression in diseased versus healthy tissues. We illustrate that expression profiles derived from whole tissue RNA highly reflect the degenerating tissues' altered cellular composition, not necessarily transcriptional regulation. To accurately understand transcriptional changes that accompany neuropathology, we acutely purify neurons, astrocytes and microglia from single adult mouse brains and analyse their transcriptomes by RNA sequencing. Using peripheral endotoxemia to establish the method, we reveal highly specific transcriptional responses and altered RNA processing in each cell type, with Tnfr1 required for the astrocytic response. Extending the method to an Alzheimer's disease model, we confirm that transcriptomic changes observed in whole tissue are driven primarily by cell type composition, not transcriptional regulation, and identify hundreds of cell type-specific changes undetected in whole tissue RNA. Applying similar methods to additional models and patient tissues will transform our understanding of aberrant gene expression in neurological disease.
Highlights
A common approach to understanding neurodegenerative disease is comparing gene expression in diseased versus healthy tissues
Most of the transcripts with increased abundance in frontotemporal dementia (FTD) cortex mapped to non-neuronal cell types— astrocytes and microglia, as well as endothelial cells—whereas most of the transcripts with decreased abundance mapped to neurons (Fig. 1a,b)
Our examination of existing data sets from neurodegenerative disease tissues revealed a common trend—that genes with decreased mRNA abundance were typically expressed by neurons, and genes with increased mRNA abundance were typically expressed by microglia or astrocytes—suggesting that many changes resulted not from transcriptional modulation but from neuronal loss and the attendant increase in glial cell fraction
Summary
A common approach to understanding neurodegenerative disease is comparing gene expression in diseased versus healthy tissues. To accurately understand transcriptional changes that accompany neuropathology, we acutely purify neurons, astrocytes and microglia from single adult mouse brains and analyse their transcriptomes by RNA sequencing. Expression profiles derived from whole tissue RNA represent each gene’s average expression among all cells but do not reveal which cell types are responsible for a gene’s normal or altered expression in healthy or diseased tissues Lacking such information, the genes and pathways implicated by profiling whole tissues cannot be readily incorporated into cellular models of neurodegenerative disease. We utilize an approach that avoids some of the abovementioned limitations[12] and adapt it further to isolate populations of neurons, astrocytes and microglia from single adult brain specimens and analyse their transcriptomes by RNA amplification and sequencing.
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