Inactivation of Hedgehog signal transduction in adult astrocytes results in region-specific blood–brain barrier defects

In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood–brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of Gli1, a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., Science 334, 1727–1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS.

Production and Purification of A Cell-Penetrating Peptide-Erythropoietin Fusion Protein and Optimization of an in Vitro Blood-Brain Barrier Model for Central Nervous System Drug Delivery

Drug delivery into the central nervous system (CNS) is a brilliant research field, and the development of protein production and purification procedures for novel therapeutic proteins is crucial. Erythropoietin (EPO) is a glycoprotein with tremendous neuroprotective potential, but its bulky size prevents easy penetration across the blood-brain barrier (BBB). EPO-HBHAc is a promising cell-penetrating peptide modified protein for CNS diseases, necessitating an appropriate in vitro BBB model for further evaluation. The plasmid of EPO-HBHAc was constructed by DNA recombinant technology, and the Chinese Hamster Ovary (CHO-K1) cell expression system was selected to generate target proteins. His-tag and size exclusion purification were used to purify the target protein from the cell-conditioned medium; target proteins were further evaluated by western blotting and Coomassie blue staining. Moreover, the endothelial cells (bEnd.3) and astrocytes (CTX TNA2) were used to generate the in vitro BBB model, and transepithelial electrical resistance (TEER) and paracellular diffusion were measured to evaluate barrier integrity. The EPO-HBHAc plasmid was successfully constructed, and a stable cell line expressing EPO-HBHAc was generated. A higher protein expression level was observed in serum-containing medium than in serum-free medium. His-tag purification is not sufficient to remove impurities from target proteins, and thus size exclusion purification was performed to increase the purity of the protein of interest. In contrast, a higher TEER value and lower paracellular diffusion were observed in the co-culture model than in the mono-culture model. Furthermore, the higher TEER value was observed in inserts with a larger growth area (4.67 cm2) than in those with a smaller area (0.33 cm2). In conclusion, we demonstrated that some critical points might impact protein production and the in vitro BBB model construction in this study. Importantly, our research will provide valuable information in the field of CNS drug delivery.

Porcine rotavirus mainly infects primary porcine enterocytes at the basolateral surface

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host’s innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.

Claudin-12 is not required for blood–brain barrier tight junction function

Background The blood–brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown. Methods We created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant. Results Making use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice. Conclusions Our present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions.

Autoregulated paracellular clearance of amyloid-β across the blood-brain barrier

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting neural tissue from damaging blood-borne agents. The barrier is characterized by endothelial tight junctions that limit passive paracellular diffusion of polar solutes and macromolecules from blood to brain. Decreased brain clearance of the neurotoxic amyloid-β (Aβ) peptide is a central event in the pathogenesis of Alzheimer’s disease (AD). Whereas transport of Aβ across the BBB can occur via transcellular endothelial receptors, the paracellular movement of Aβ has not been described. We show that soluble human Aβ(1–40) monomers can diffuse across the paracellular pathway of the BBB in tandem with a decrease in the tight junction proteins claudin-5 and occludin in the cerebral vascular endothelium. In a murine model of AD (Tg2576), plasma Aβ(1–40) levels were significantly increased, brain Aβ(1–40) levels were decreased, and cognitive function was enhanced when both claudin-5 and occludin were suppressed. Furthermore, Aβ can cause a transient down-regulation of claudin-5 and occludin, allowing for its own paracellular clearance across the BBB. Our results show, for the first time, the involvement of the paracellular pathway in autoregulated Aβ movement across the BBB and identify both claudin-5 and occludin as potential therapeutic targets for AD. These findings also indicate that controlled modulation of tight junction components at the BBB can enhance the clearance of Aβ from the brain.

Brain barriers: Crosstalk between complex tight junctions and adherens junctions

Unique intercellular junctional complexes between the central nervous system (CNS) microvascular endothelial cells and the choroid plexus epithelial cells form the endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier (BCSFB), respectively. These barriers inhibit paracellular diffusion, thereby protecting the CNS from fluctuations in the blood. Studies of brain barrier integrity during development, normal physiology, and disease have focused on BBB and BCSFB tight junctions but not the corresponding endothelial and epithelial adherens junctions. The crosstalk between adherens junctions and tight junctions in maintaining barrier integrity is an understudied area that may represent a promising target for influencing brain barrier function.

An in vitro investigation of species-dependent intestinal transport of selenium and the impact of this process on selenium bioavailability

A range of Se species has been shown to occur in a variety of different foodstuffs. Depending on its speciation, Se is more or less bioavailable to human subjects. In the present study, the role of speciation as a determinant of Se bioavailability was addressed with an investigation of species-specific mechanisms of transport at the intestinal level. The present work focused on four distinct Se compounds (selenate (Se(VI)), selenite (Se(IV)), selenomethionine (SeMet) and methylselenocysteine (MeSeCys)), whose intestinal transport was mimicked through an in vitro bicameral model of enterocyte-like differentiated Caco-2 cells. Efficiency of Se absorption was shown to be species dependent (SeMet>MeSeCys>Se(VI)>Se(IV)). In the case of SeMet, MeSeCys and Se(VI), the highly polarised passage from the apical to basolateral pole indicated that a substantial fraction of transport was transcellular, whilst results for Se(IV) indicated paracellular diffusion. Passage of the organic Se species (SeMet and MeSeCys) became saturated after 3 h, but no such effect was observed for the inorganic species. In addition, SeMet and MeSeCys transport was significantly inhibited by their respective S analogues methionine and methylcysteine, which suggests a common transport system for both kinds of compounds.

RalA and RalB differentially regulate development of epithelial tight junctions

Tight junctions (TJs) are structures indispensable to epithelial cells and are responsible for regulation of paracellular diffusion and maintenance of cellular polarity. Although many interactions between TJ constituents have been identified, questions remain concerning how specific functions of TJs are established and regulated. Here we investigated the roles of Ral GTPases and their common effector exocyst complex in the formation of nascent TJs. Unexpectedly, RNA interference–mediated suppression of RalA or RalB caused opposing changes in TJ development. RalA reduction increased paracellular permeability and decreased incorporation of components into TJs, whereas RalB reduction decreased paracellular permeability and increased incorporation of components into TJs. Activities of both Ral GTPases were mediated through the exocyst. Finally, we show that TJ-mediated separation of apical–basal membrane domains is established prior to equilibration of barrier function and that it is unaffected by Ral knockdown or specific composition of TJs.