The low level of transcytosis is a unique feature of cerebrovascular endothelial cells (ECs), ensuring restrictive blood-brain barrier (BBB) permeability. Major facilitator superfamily domain-containing 2a (MFSD2A) is a key regulator of the BBB function by suppressing caveolae-mediated transcytosis. However, the mechanisms regulating MFSD2A at the BBB have been barely explored. Here, we show that cerebrovascular EC-specific deletion of Pten (phosphatase and tensin homolog) results in a dramatic increase in vesicular transcytosis by the reduction of MFSD2A, leading to increased transcellular permeability of the BBB. Mechanistically, AKT signaling inhibits E3 ubiquitin ligase NEDD4-2-mediated MFSD2A degradation. Consistently, cerebrovascular Nedd4-2 overexpression decreases MFSD2A levels, increases transcytosis, and impairs BBB permeability, recapitulating the phenotypes of Pten-deficient mice. Furthermore, Akt deletion decreases phosphorylated NEDD4-2 levels, restores MFSD2A levels, and normalizes BBB permeability in Pten-mutant mice. Altogether, our work reveals the essential physiological function of the PTEN/AKT/NEDD4-2/MFSD2A axis in the regulation of BBB permeability.

Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2^Cas9). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2^Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs), a key component of the endosomal sorting complex required for transport (ESCRT), has been implicated in many essential biological processes. However, the physiological role of endogenous Hgs in the vascular system has not previously been explored. Here, we have generated brain endothelial cell (EC) specific Hgs knockout mice to uncover the function of Hgs in EC polarity and cerebrovascular stability. Knockout of Hgs in brain ECs led to impaired endothelial apicobasal polarity and brain vessel collapse in mice. We determined that Hgs is essential for recycling of vascular endothelial (VE)-cadherin to the plasma membrane, since loss of Hgs blocked trafficking of endocytosed VE-cadherin from early endosomes to recycling endosomes, and impaired the motility of recycling endosomes. Supportively, overexpression of the motor kinesin family member 13A (KIF13A) restored endosomal recycling and rescued abrogated polarized trafficking and distribution of VE-cadherin in Hgs knockdown ECs. These data uncover a novel physiological function of Hgs and support an essential role for the ESCRT machinery in the maintenance of EC polarity and cerebrovascular stability.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood–brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26^LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

This study proposes a novel occupational therapy (OT) practice tool derived from the native concept of the Tai Chi diagram. Four elements of the Tai Chi diagram, namely, Yang, Yin, Yang kernel, and Yin kernel, are matched to the elements of the OT practice, namely, person, environment, economic condition, and mental condition, respectively. The entire circle of the diagram represents occupational performance. The interaction between persons and the environment can be regarded as the interaction between Yin and Yang with balance as the focal point. This proposed tool may be an innovative means to facilitate communication between clients and therapists for OT in China.