African trypanosomes and brain infection – the unsolved question

African trypanosomes induce sleeping sickness. The parasites are transmitted during the blood meal of a tsetse fly and appear primarily in blood and lymph vessels, before they enter the central nervous system. During the latter stage, trypanosomes induce a deregulation of sleep–wake cycles and some additional neurological disorders. Historically, it was assumed that trypanosomes cross the blood–brain barrier and settle somewhere between the brain cells. The brain, however, is a strictly controlled and immune‐privileged area that is completely surrounded by a dense barrier that covers the blood vessels: this is the blood–brain barrier. It is known that some immune cells are able to cross this barrier, but this requires a sophisticated mechanism and highly specific cell–cell interactions that have not been observed for trypanosomes within the mammalian host. Interestingly, trypanosomes injected directly into the brain parenchyma did not induce an infection. Likewise, after an intraperitoneal infection of rats, Trypanosoma brucei brucei was not observed within the brain, but appeared readily within the cerebrospinal fluid (CSF) and the meninges. Therefore, the parasite did not cross the blood–brain barrier, but the blood–CSF barrier, which is formed by the choroid plexus, i.e. the part of the ventricles where CSF is produced from blood. While there is no question that trypanosomes are able to invade the brain to induce a deadly encephalopathy, controversy exists about the pathway involved. This review lists experimental results that support crossing of the blood–brain barrier and of the blood–CSF barrier and discuss the implications that either pathway would have on infection progress and on the survival strategy of the parasite. For reasons discussed below, we prefer the latter pathway and suggest the existence of an additional distinct meningeal stage, from which trypanosomes could invade the brain via the Virchow–Robin space thereby bypassing the blood–brain barrier. We also consider healthy carriers, i.e. people living symptomless with the disease for up to several decades, and discuss implications the proposed meningeal stage would have for new anti‐trypanosomal drug development. Considering the re‐infection of blood, a process called relapse, we discuss the likely involvement of the newly described glymphatic connection between the meningeal space and the lymphatic system, that seems also be important for other infectious diseases.     

Pathogen translocation across the blood–brain barrier

Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood–brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia ) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial–host interactions (receptor–ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo . This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.     

Fetuin‐A in the developing brain

The serum protein fetuin‐A is essential for mineral homeostasis and shows immunomodulatory functions, for example by binding to TGF superfamily proteins. It proved neuroprotective in a rat stroke model and reduced lethality after systemic lipopolysaccharide challenge in mice. Serum fetuin‐A concentrations are highest during intrauterine life. Different species show intrauterine cerebral fetuin‐A immunoreactivity, suggesting a contribution to brain development. We therefore aimed at specifying fetuin‐A immunoreactivity in brains of newborn rats (age P0–P28) and human neonates (20–40 weeks of gestation). In humans and rats, fetuin‐A was found in cortex, white matter, subplate, hippocampus, subventricular zone, and ependymal cells which supports a global role for brain function. In rats, overall fetuin‐A immunoreactivity decreased with age. At P0 fetuin‐A immunoreactivity affected most brain structures. Thereafter, it became increasingly restricted to distinct cells of the hippocampus, cingular gyrus, periventricular stem cell layer, and ependyma. In ependymal cells the staining pattern complied with active transependymal transport from cerebrospinal fluid. Double immunofluorescence studies revealed colocalization with NeuN (mature neurons), beta III tubulin (immature neurons), GFAP (astrocytes), and CD68 (activated microglia). This points to a role of fetuin‐A in different brain functional systems. In human neonatal autopsy cases, frequently affected from severe neurological and non‐neurological diseases, fetuin‐A immunoreactivity was heterogeneous and much less associated with age than in healthy tissues studied earlier, suggesting an impact of exogeneous noxious factors on fetuin‐A regulation. Further research on the role of fetuin‐A in the neonatal brain during physiological and pathological conditions is recommended. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 354–369, 2013     

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

The blood–brain barrier (BBB ) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make‐up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15‐fold enrichment of endothelial cell marker Glut1 mRNA , whereas markers for other cell types were not enriched. Filter‐aided sample preparation was shown to be superior to in‐solution sample preparation (10251 peptides vs. 7533 peptides). Label‐free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP‐binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

     

Dl‐3n‐butylphthalide improves traumatic brain injury recovery via inhibiting autophagy‐induced blood‐brain barrier disruption and cell apoptosis

Blood‐brain barrier (BBB) disruption and neuronal apoptosis are important pathophysiological processes after traumatic brain injury (TBI). In clinical stroke, Dl‐3n‐butylphthalide (Dl‐NBP) has a neuroprotective effect with anti‐inflammatory, anti‐oxidative, anti‐apoptotic and mitochondrion‐protective functions. However, the effect and molecular mechanism of Dl‐NBP for TBI need to be further investigated. Here, we had used an animal model of TBI and SH‐SY5Y/human brain microvascular endothelial cells to explore it. We found that Dl‐NBP administration exerts a neuroprotective effect in TBI/OGD and BBB disorder, which up‐regulates the expression of tight junction proteins and promotes neuronal survival via inhibiting mitochondrial apoptosis. The expressions of autophagy‐related proteins, including ATG7, Beclin1 and LC3II, were significantly increased after TBI/OGD, and which were reversed by Dl‐NBP treatment both in vivo and in vitro. Moreover, rapamycin treatment had abolished the effect of Dl‐NBP for TBI recovery. Collectively, our current studies indicate that Dl‐NBP treatment improved locomotor functional recovery after TBI by inhibiting the activation of autophagy and consequently blocking the junction protein loss and neuronal apoptosis. Dl‐NBP, as an anti‐inflammatory and anti‐oxidative drug, may act as an effective strategy for TBI recovery.     

Staphylococcus aureus‐mediated blood–brain barrier injury: an in vitro human brain microvascular endothelial cell model

Blood–brain barrier (BBB) disruption constitutes a hallmark event during pathogen‐mediated neurological disorders such as bacterial meningitis. As a prevalent opportunistic pathogen, Staphylococcus aureus (SA) is of particular interest in this context, although our fundamental understanding of how SA disrupts the BBB is very limited. This paper employs in vitro infection models to address this. Human brain microvascular endothelial cells (HBMvECs) were infected with formaldehyde‐fixed (multiplicity of infection [MOI] 0–250, 0–48 hr) and live (MOI 0–100, 0–3 hr) SA cultures. Both Fixed‐SA and Live‐SA could adhere to HBMvECs with equal efficacy and cause elevated paracellular permeability. In further studies employing Fixed‐SA, infection of HBMvECs caused dose‐dependent release of cytokines/chemokines (TNF‐α, IL‐6, MCP‐1, IP‐10, and thrombomodulin), reduced expression of interendothelial junction proteins (VE‐Cadherin, claudin‐5, and ZO‐1), and activation of both canonical and non‐canonical NF‐κB pathways. Using N‐acetylcysteine, we determined that these events were coupled to the SA‐mediated induction of reactive oxygen species (ROS) within HBMvECs. Finally, treatment of HBMvECs with Fixed‐ΔSpA (MOI 0–250, 48 hr), a gene deletion mutant of Staphylococcal protein A associated with bacterial infectivity, had relatively similar effects to Newman WT Fixed‐SA. In conclusion, these findings provide insight into how SA infection may activate proinflammatory mechanisms within the brain microvascular endothelium to elicit BBB failure.     

Glial‐cell‐mediated re‐induction of the blood‐brain barrier phenotype in brain capillary endothelial cells: A differential gel electrophoresis study

In the neurovascular unit, brain microvascular endothelial cells develop characteristic barrier features that control the molecular exchanges between the blood and the brain. These characteristics are partially or totally lost when the cells are isolated for use in in vitro blood‐brain barrier (BBB) models. Hence, the re‐induction of barrier properties is crucial for the relevance of BBB models. Although the role of astrocyte promiscuity is well established, the molecular mechanisms of re‐induction remain largely unknown. Here, we used a DIGE‐based proteomics approach to study endothelial cellular proteins showing significant quantitative variations after BBB re‐induction. We confirm that quantitative changes mainly concern proteins involved in cell structure and motility. Furthermore, we describe the possible involvement of the asymmetric dimethylarginine pathway in the BBB phenotype re‐induction process and we discuss asymmetric dimethylarginine's potential role in regulating endothelial function (in addition to its role as a by‐product of protein modification). Our results also suggest that the intracellular redox potential is lower in the in vitro brain capillary endothelial cells displaying re‐induced BBB functions than in cells with limited BBB functions.