RESUMEN
Single-nucleus RNA sequencing (snRNA-seq) is often used to define gene expression patterns characteristic of brain cell types as well as to identify cell type specific gene expression signatures of neurological and mental illnesses in postmortem human brains. As methods to obtain brain tissue from living individuals emerge, it is essential to characterize gene expression differences associated with tissue originating from either living or postmortem subjects using snRNA-seq, and to assess whether and how such differences may impact snRNA-seq studies of brain tissue. To address this, human prefrontal cortex single nuclei gene expression was generated and compared between 31 samples from living individuals and 21 postmortem samples. The same cell types were consistently identified in living and postmortem nuclei, though for each cell type, a large proportion of genes were differentially expressed between samples from postmortem and living individuals. Notably, estimation of cell type proportions by cell type deconvolution of pseudo-bulk data was found to be more accurate in samples from living individuals. To allow for future integration of living and postmortem brain gene expression, a model was developed that quantifies from gene expression data the probability a human brain tissue sample was obtained postmortem. These probabilities are established as a means to statistically account for the gene expression differences between samples from living and postmortem individuals. Together, the results presented here provide a deep characterization of both differences between snRNA-seq derived from samples from living and postmortem individuals, as well as qualify and account for their effect on common analyses performed on this type of data.
RESUMEN
Adenosine-to-inosine (A-to-I) editing is a prevalent post-transcriptional RNA modification within the brain. Yet, most research has relied on postmortem samples, assuming it is an accurate representation of RNA biology in the living brain. We challenge this assumption by comparing A-to-I editing between postmortem and living prefrontal cortical tissues. Major differences were found, with over 70,000 A-to-I sites showing higher editing levels in postmortem tissues. Increased A-to-I editing in postmortem tissues is linked to higher ADAR1 and ADARB1 expression, is more pronounced in non-neuronal cells, and indicative of postmortem activation of inflammation and hypoxia. Higher A-to-I editing in living tissues marks sites that are evolutionarily preserved, synaptic, developmentally timed, and disrupted in neurological conditions. Common genetic variants were also found to differentially affect A-to-I editing levels in living versus postmortem tissues. Collectively, these discoveries illuminate the nuanced functions and intricate regulatory mechanisms of RNA editing within the human brain.
RESUMEN
A goal of medical research is to determine the molecular basis of human brain health and illness. One way to achieve this goal is through observational studies of gene expression in human brain tissue. Due to the unavailability of brain tissue from living people, most such studies are performed using tissue from postmortem brain donors. An assumption underlying this practice is that gene expression in the postmortem human brain is an accurate representation of gene expression in the living human brain. Here, this assumption - which, until now, had not been adequately tested - is tested by comparing human prefrontal cortex gene expression between 275 living samples and 243 postmortem samples. Expression levels differed significantly for nearly 80% of genes, and a systematic examination of alternative explanations for this observation determined that these differences are not a consequence of cell type composition, RNA quality, postmortem interval, age, medication, morbidity, symptom severity, tissue pathology, sample handling, batch effects, or computational methods utilized. Analyses integrating the data generated for this study with data from earlier landmark studies that used tissue from postmortem brain donors showed that postmortem brain gene expression signatures of neurological and mental illnesses, as well as of normal traits such as aging, may not be accurate representations of these gene expression signatures in the living brain. By using tissue from large cohorts living people, future observational studies of human brain biology have the potential to (1) determine the medical research questions that can be addressed using postmortem tissue as a proxy for living tissue and (2) expand the scope of medical research to include questions about the molecular basis of human brain health and illness that can only be addressed in living people (e.g., "What happens at the molecular level in the brain as a person experiences an emotion?").
