The secreted neuronal signal Spock1 promotes blood-brain barrier development

Summary

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restrict its permeability to proteins and small molecules. Classic chick-quail chimera studies have shown that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here, we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1 partially restores BBB function in spock1 mutants by quenching gelatinase activity and restoring vascular expression of BBB genes including mcamb. These analyses support a model in which neuronally secreted Spock1 initiates BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression.

Introduction

The blood-brain barrier (BBB) maintains a tightly controlled homeostatic environment in the brain that is required for proper neural function. BBB breakdown has been implicated in multiple neurodegenerative diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases.¹ Conversely, the BBB also serves as an obstacle for effective drug delivery to the brain. Therefore, there is great interest in generating a better understanding of how to therapeutically regulate its permeability, both to restore BBB function in neurodegeneration and to transiently open it for improved chemotherapeutic access for brain tumors. The BBB is a specialized property of the brain vasculature, which is composed of a thin, continuous layer of non-fenestrated endothelial cells with uniquely restrictive properties. Brain endothelial cells create the barrier via two primary cellular mechanisms: (1) specialized tight junction complexes that block the transit of small water-soluble molecules between cells and (2) reduced levels of vesicular trafficking or transcytosis to restrict transit through endothelial cells.² At a molecular level, functional barrier endothelial cells are differentiated from peripheral endothelial cells by high expression of the tight junction molecule Claudin-5³ and the fatty acid transporter Mfsd2a,^4,5 and an absence of the fenestra and vesicle-associated protein PLVAP.⁶ Expression of substrate-specific influx and efflux transporters such as Glut1⁷ and Pgp,⁸ which dynamically regulate the intake of necessary nutrients and the removal of metabolic waste products,⁹ further enhances the selectivity of the BBB.

The restrictive properties of the BBB are not intrinsic to brain endothelial cells but are induced and maintained by signals in the brain microenvironment during embryonic development.¹⁰ When avascularized quail neural tissue was transplanted into embryonic chicken gut cavities, the blood vessels ingressing from the non-barrier gut tissue obtained functional BBB properties, specifically both tight junctions and reduced levels of vesicles, resulting in trypan blue tracer confinement within the blood vessels.¹⁰ Conversely, when avascularized quail somite tissue was transplanted into embryonic chick brain, the blood vessels ingressing from the neural tissue lost functional BBB properties, leaking tracer into the quail graft due to increased endothelial transcytosis and loss of tight junction function. These data show that signals from the neural microenvironment to the vasculature control barrier function. Furthermore, these microenvironmental signals are also required to actively maintain barrier properties throughout life.^11,12,13,14

Mural cells called pericytes share the endothelial basement membrane and co-migrate with endothelial cells during neovascularization of the developing brain. This close interaction between pericytes and endothelial cells is required for both the initiation and continued maintenance of barrier properties.^15,16,17 Astrocytes, glial cells found exclusively in the central nervous system (CNS), completely ensheath the vasculature with their endfeet in a polarized fashion.¹⁸ Astrocytes arise late in embryonic development, after neurogenesis is complete, with the vast majority of gliogenesis occurring postnatally in rodents. Mammalian astrocytes are potent inducers of barrier properties in vitro and in vivo and are required for BBB maintenance.^13,14,19 While the necessity of these two cell types has been known for decades, canonical Wnt signaling arising from both neuronal and astrocytic sources is the only microenvironmental signal known to induce and maintain BBB function.^{13,20,21,22,23,24,25,26,27} However, in addition to its role in regulating barrier properties, Wnt signaling also plays an important role in tip cell specification and angiogenesis in the developing brain.^21,23,24 Furthermore, mouse endothelial cells are Wnt responsive as early as embryonic day (E) 9.5,²⁷ preceding the acquisition of functional barrier properties,²² suggesting that other signaling may be acting in conjunction with or in addition to Wnt signaling for the induction of BBB properties.

To further examine the molecular determinants of BBB development, we turned to the optically transparent zebrafish system, which allows for imaging of the intact BBB in toto throughout zebrafish development. Zebrafish brain endothelial cells express many of the same molecular markers as mammalian brain endothelial cells, including Glut1, Cldn5, ZO-1, and Mfsd2a.^{24,28,29,30,31,32} We previously characterized the molecular and subcellular mechanisms of functional BBB development in zebrafish and determined that the zebrafish BBB becomes functionally mature by 5 days post fertilization (dpf) due to the suppression of transcytosis rather than through the acquisition of tight junction function, which was observed as early as 3 dpf.²⁸ During these studies, we serendipitously discovered a recessive viable mutant with profound forebrain and midbrain barrier leakage, which we identify here to be the neuronally produced and secreted proteoglycan Spock1. Using an array of perturbations and imaging techniques, we uncover the range of Spock1 signaling to the vasculature and identify that mechanistically, Spock1 signaling acts through control of the pericyte-endothelial extracellular matrix (ECM) to control endothelial transcytosis…