(W1030-05-31) Pericyte Control of Gene Expression in the Brain Endothelium: Implications for Alzheimer’s disease

Purpose: Late-onset Alzheimer’s disease (AD) is the major cause of dementia in the elderly. A strong body of evidence suggests that changes in the brain vasculature associated with AD are not simply features of the disease, but among its driving forces. One distinctive feature of this cerebrovascular dysfunction is the degeneration of brain pericytes -- often overlooked supporting cells of blood-brain barrier endothelium. The endothelial cell monolayer, sealed by paracellular tight junctions, forms the bulwark of the blood-brain barrier (BBB), which tightly regulates solute exchange between blood and brain. Pericytes, which wrap around the abluminal side of the BBB endothelium, are the main supporting cells of the BBB. Pericytes regulate blood flow by controlling microvascular dilation and play a critical role in the development, maintenance, and homeostatic function of the BBB. Pericytes are also believed to regulate amyloid-β clearance, glucose transport, immune response, and inflammation, and several studies have linked pericyte degeneration with the acceleration of amyloid-β peptide deposition, abnormal tau protein accumulation, and neurodegeneration in AD. Very little is known, however, about the molecular mechanisms and biological pathway disruptions involved in pericyte degeneration. In this study, we investigated the effect of pericyte degeneration on the BBB by characterizing pericyte control of gene and protein expression in the BBB endothelium. Two aspects of AD pathology under investigation are specifically relevant: disruption of insulin signaling in the brain, including the in BBB endothelium, and cerebral iron accumulation, both of which have been shown to contribute to AD amyloidiosis, oxidative stress, inflammation, and cognitive decline. Excess accumulation of intercellular iron is the driver of ferroptosis, an iron-induced cell death pathway increasingly associated with AD. IGF2, BDNF and activin A, are secreted proteins that are believed to have synergistic, neurotrophic, and neuroprotective properties. Pericytes could alter endothelial expression and BBB transport of these proteins to the brain parenchyma.

Methods: Bioinformatic analysis was conducted on an RNA-Seq expression counts matrix obtained from the Gene Expression Omnibus database (GSE144474)1, which compared human stem-cell derived endothelial cells (ECs) cultured solo against ECs co-cultured with immortalized human brain pericytes in a non-contact Transwell model over 96 hours. We replicated the Transwell model with primary human brain pericytes and measured both RNA and protein expression with reverse transcription quantitative real-time PCR (RT-qPCR) and Western blots, respectively.

Results: PCA, permutation, Venn, and alluvial analysis demonstrate that all endothelial solo cultures are distinct from endothelial-pericyte cocultures and exhibit relative equilibrium. In contrast, pericyte-endothelial cocultures display longitudinal clustering. A comparison of differential expression profiles among the cocultures suggest an initial perturbation of gene expression at 24 hours, followed by a movement toward a new equilibrium. Results from Short Time-series Expression Miner (STEM) show a statistical bias toward dynamic expression profiles exhibiting biologically meaningful patterns. Pathway enrichment analysis was performed on KEGG gene sets using both over-representation and GSEA methods. The insulin resistance and ferroptosis pathways were enriched at all time points. Other time-specific enriched pathways included downregulation of pathways promoting cell death, inflammation, and proliferation, as well as upregulation of pathways involved in extracellular matrix formation and protein translation. Among the most enriched and important genes affecting long-term insulin signaling and ferroptosis include the insulin receptor (INSR), suppressor of cytokine signaling 3 (SOCS3), Glut 1 (SLC2A1), heme oxygenase 1 (HMOX1), ferritin (FTL, FTH1), and transferrin (TFRC). Western blots were conducted on INSR and HMOX1, which were concordant with RNA-Seq data. Additionally, several genes relevant to AD pathology were upregulated more than 5-fold, including insulin growth factor 2 (IGF2), brain-derived neurotrophic factor (BDNF) and inhibin A (INHBA), which codes for the homodimer activin A.

Conclusion: Pericyte influence on endothelial insulin signaling involves a complex system of signal amplifiers, negative regulators, and feedback. Taken together, the picture suggests a more robust equilibrium, as well as protection from insulin resistance and AD amyloidosis. In addition, pericyte regulation of genes involved in insulin signaling include several kinases believed to protect against AD pathology by regulating inflammation, oxidative stress, BBB function, microvessel integrity, neuroprotection and glucose transport. Endothelial solo cultures lacking pericytes also exhibit dysregulated iron homeostasis consistent with a tendency toward ferroptosis, and pericytes were shown to upregulate secreted neurotrophic and neuroprotective proteins. In conclusion, pericyte degeneration disrupts key pathways associated with AD pathophysiology that likely contribute to disease progression and alert us to therapeutic targets.

References: 1. Marjolein Heymans, Ricardo Figueiredo, Lucie Dehouck, David Francisco, Yasuteru Sano, Fumitaka Shimizu, Takashi Kanda, R ́emy Bruggmann, Britta Engelhardt, Peter Winter, Fabien Gosselet, and Maxime Culot. Contribution of brain pericytes in blood–brain barrier formation and maintenance: a transcriptomic study of cocultured human endothelial cells derived from hematopoietic stem cells. Fluids and Barriers of the CNS, 17(1):48, December 2020.

Acknowledgements: This work was supported by the Minnesota Partnership for Biotechnology and Medical Genomics (MNP#15.31) and National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS125437).

Enriched pathways using overrepresentation (DAVID) and Gene Set Enrichment analysis (GSEA). Values in the table represent −sgn(dir) · log10(pvalue), where sgn(dir) is positive for upregulated pathways and negative for downregulated pathways. Thus, red indicates upregulation and blue downregulation. All listed pathways are enriched at some time based on at least one of the two methods. Enrichment is defined as having both − log10(pvalue) > 2 along with an FDR of less than 0.05 in the case of DAVID and 0.20 for GSEA. The left table includes all pathways enriched at 96 hours, while the table on the right includes pathways enriched at other times.

Differentially expressed genes in the insulin resistance (a,b,c) and ferroptosis (d) pathways. Included are all genes in each pathway with FDR < 0.01.

Western Blot for insulin receptor β subunit protein (IR-β) and heme oxygenase 1 (HO-1). At left is gel with vinculin loading control. Bar plots of IR-β and HO-1 show protein absorbance normalized to loading control. Y-axis scale is relative to mean of endothelial solo culture absorbance. P-values for IR-β and HO-1 were 0.0348 and 0.0068, respectively.