The Human Blood-Nerve Barrier Transcriptome

Abstract

The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment essential for effective axonal signal transduction. Relatively little is known about the unique human BNB molecular composition. Such knowledge is crucial to comprehend the relationships between the systemic circulation and peripheral nerves in health, adaptations to intrinsic or extrinsic perturbations and alterations that may result in disease. We performed RNA-sequencing on cultured early- and late-passage adult primary human endoneurial endothelial cells and laser-capture microdissected endoneurial microvessels from four cryopreserved normal adult human sural nerves referenced to the Genome Reference Consortium Human Reference 37 genome browser, using predefined criteria guided by known transcript or protein expression in vitro and in situ. We identified 12881 common transcripts associated by 125 independent biological networks, defined as the normal adult BNB transcriptome, including a comprehensive array of transporters and specialized intercellular junctional complex components. These identified transcripts and their interacting networks provide insights into peripheral nerve microvascular morphogenesis, restrictive barrier formation, influx and efflux transporters with relevance to understanding peripheral nerve homeostasis and pharmacology, including targeted drug delivery and the mediators of leukocyte trafficking in peripheral nerves during normal immunosurveillance.

In situ molecular characterization of endoneurial microvessels that form the blood‐nerve barrier in normal human adult peripheral nerves

“…Indirect fluorescent immunohistochemistry demonstrated protein expression in ≥90% of the endoneurial microvessels identified in the four adult sural nerve biopsies (range 90%‐100%), verifying uniform in situ expression of the selected proteins consistent with the previously published transcriptome data (Table ). The mean in situ transcript expression levels in laser capture microdissected endoneurial microvessels for each gene studied (based on fragments per kilobase of fragments per million) has been previously published . Sequencing data have also been published and are freely accessible via the National Center for Biotechnology Information Gene Expression Omnibus series accession number GSE107574: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107574…”

“…ABCB1 (also known as P‐glycoprotein, a major efflux transporter) and SLC1A1 (also known as excitatory amino acid transporter‐3 that actively transports glutamate across plasma membranes) were the only proteins restricted to endoneurial microvessel endothelium (Figure ), implying crucial roles in normal human BNB function.…”

“…It is important to recognize that most of the proteins studied are expressed outside of the BNB in peripheral nerves, and that “classic” markers of vascular endothelial tight and adherens junctions, CLDN5 and CDH5, were not restricted to human adult endoneurial microvascular endothelium in situ, emphasizing the need for further study to elucidate the specific molecular components of the uniquely restrictive human adult BNB intercellular junctional complex and its specialized transporters. Guided by the extensive transcript data present in the human BNB transcriptome, comparative in situ studies with the human blood‐brain barrier are feasible so as to ascertain unique human BNB characteristics.…”

“…Axial cryostat sections were mounted on Superfrost Plus microscope slides (catalog # 4951PLUS4; Thermo Fisher Scientific) and maintained at −20°C prior to processing. Twenty‐five proteins, including junctional complex (tight, adherens, gap), specialized transporters, cytoskeletal, and cell membrane proteins were selected for investigation, guided by the recently elucidated human BNB transcriptome …”

“…Claudin‐5 (CLDN5), zona occludens‐1 (ZO‐1, also known as tight junction protein‐1 [TJP1]), and the vascular‐specific cadherin‐5 (CDH5, also known as vascular endothelial [VE]‐cadherin), are commonly cited as molecular components of the specialized BNB junctional complex, with alterations implicated in the pathogenesis of specific peripheral neuropathies . However, data have emerged over the past decade on the complexity of specialized intercellular tight and adherens junctional complexes formed by specific claudins and cadherins interacting with adapter molecules such members of the ZO‐1 subfamily, catenins, and other membrane‐associated junctional proteins that bind directly or indirectly to cytoskeletal proteins and are responsible for the cellular structural integrity, polarity, and the unique biologic functions during normal physiological states …”