Single-nucleus RNA sequencing of the midbrain blood-brain barrier cells in schizophrenia

Abstract

Background: The increased inflammatory state observed in schizophrenia post-mortem brain and midbrain tissue [1-5] may have detrimental consequences on the brain vasculature of patients with schizophrenia. On the other hand, intrinsic alterations in the brain vasculature of schizophrenia patients may impair toxin efflux and favour the ingression of toxic material and immune cells into the central nervous system, leading to neuroinflammation. Evidence derived from blood and cerebrospinal fluid measurements indicated increased levels of adhesion molecules, such as sP-selectin and sICAM, in schizophrenia [6-8], which may favour the ingression of immune cells into the brain, and higher levels of albumin in CSF as compared to controls, suggesting increased BBB permeability in schizophrenia patients [6, 7, 9]. However, the proper functioning of the BBB involves a variety of cell types, and it is not known which cell type(s) of the BBB may be particularly affected in schizophrenia. Methods: We combined a fluorescence activated sorting isolation strategy with single-nucleus RNA sequencing (snRNAseq) to characterize the cells of the BBB in the midbrain of 15 schizophrenia donors and 14 matched controls, from the well-characterized Stanley Brain Collection. The cases used for the present study were previously grouped into high and low inflammation cases, based on the expression of inflammatory cytokines in their cortical grey matter, and 4 out of the 15 schizophrenia cases were assigned to the cortical high-inflammation group [10]. The different cell types of the BBB were identified with unbiased cluster analysis of nuclear transcriptomic profiles and annotated based on the expression of cell type-specific marker genes. Subclustering analysis, gene set enrichment and comparisons with previously published datasets were carried out to identify sub-populations among the main BBB cell types. We compared the transcriptomic profiles of the major BBB cell types between schizophrenia and controls. In addition, the relative abundance of the main BBB cell types and sub-populations was compared between the diagnoses, using generalized linear modeling. Results: We did not identify changes in the relative abundance of the major BBB cell types, nor in the sub-populations, associated with schizophrenia. However, we identified 14 differentially expressed genes in the cells of the BBB in schizophrenia as compared to controls, including genes that have previously been related to schizophrenia, such as FOXP2 and PDE4D. These transcriptional changes associated with schizophrenia were limited to the ependymal cells and pericytes. Additionally, we detected a sub-population of protoplasmic astrocytes enriched in the high inflammation schizophrenia subgroup. Genes more abundantly expressed in these schizophrenia-related protoplasmic astrocytes were associated with glutamatergic synaptic function rather than with inflammation. Conclusion: Our overall diagnostic results suggest the relative abundance of the BBB cell types and cellular sub-populations remains unaltered in schizophrenia pathology. Nonetheless, transcriptional changes in the midbrain BBB cells associated with schizophrenia are found, but are limited and are specific to two cell types, the ependymal and pericytes. In addition, the high inflammatory status observed in a subgroup of the patients may be interfering with the normal activities of protoplasmic astrocytes, possibly associated with midbrain dopamine and glutamate neurons.