Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Improved Method for the Establishment of an <em>In Vitro</em> Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Aarhus University

The IFNL4 gene is a recently discovered type III interferon, which in a significant fraction of the human population harbours a frameshift mutation abolishing the IFNλ4 ORF. The expression of IFNλ4 is correlated with both poor spontaneous clearance of hepatitis C virus (HCV) and poor response to treatment with type I interferon. Here, we show that the IFNL4 gene encodes an active type III interferon, named IFNλ4, which signals through the IFNλR1 and IL-10R2 receptor chains. Recombinant IFNλ4 is antiviral against both HCV and coronaviruses at levels comparable to IFNλ3. However, the secretion of IFNλ4 is impaired compared to that of IFNλ3, and this impairment is not due to a weak signal peptide, which was previously believed. We found that IFNλ4 gets N-linked glycosylated and that this glycosylation is required for secretion. Nevertheless, this glycosylation is not required for activity. Together, these findings result in the paradox that IFNλ4 is strongly antiviral but a disadvantage during HCV infection.

Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses.

Transport between the endoplasmatic reticulum, the Golgi-network, the endo-lysosomal system and the cell surface can be categorized as anterograde or retrograde, describing traffic that goes forward or backward, respectively. Traffic going from the plasma membrane to endosomes and lysosomes or the trans-Golgi network (TGN) constitutes the major retrograde transport routes. Several transmembrane proteins undergo retrograde transport as part of a recycling mechanism that contributes to reutilization and maintenance of a steady-state protein localization. In addition, some receptors are hijacked by exotoxins and used for entry and intracellular transport. The physiological relevance of retrograde transport cannot be overstated. Retrograde trafficking of the amyloid precursor protein determines the distribution between organelles, and hence the possibility of cleavage by γ-secretase. Right balancing of the pathways is critical for protection against Alzheimer's disease. During embryonic development, retrograde transport of Wntless to the TGN is essential for the following release of Wnt from the plasma membrane. Furthermore, overexpression of Wntless has been linked to oncogenesis. Here, we review relevant aspects of the retrograde trafficking of mammalian transmembrane receptors and transporters, with focus on the retromer-mediated transport between endosomes and the TGN.

Retromer-Mediated Trafficking of Transmembrane Receptors and Transporters.

The mechanisms for iron transport through the blood-brain barrier (BBB) remain a controversy. We analyzed for expression of mRNA and proteins involved in oxidation and transport of iron in isolated brain capillaries from dietary normal, iron-deficient, and iron-reverted rats. The expression was also investigated in isolated rat brain endothelial cells (RBECs) and in immortalized rat brain endothelial (RBE4) cells grown as monoculture or in hanging culture inserts with defined BBB properties. Transferrin receptor 1, ferrireductases Steap 2 and 3, divalent metal transporter 1 (DMT1), ferroportin, soluble and glycosylphosphatidylinositol (GPI)-anchored ceruloplasmin, and hephaestin were all expressed in brain capillaries in vivo and in isolated RBECs and RBE4 cells. Gene expression of DMT1, ferroportin, and soluble and GPI-anchored ceruloplasmin were significantly higher in isolated RBECs with induced BBB properties. Primary pericytes and astrocytes both expressed ceruloplasmin and hephaestin, and RBECs, pericytes, and astrocytes all exhibited ferrous oxidase activity. The coherent protein expression of these genes was demonstrated by immunocytochemistry. The data show that brain endothelial cells provide the machinery for receptor-mediated uptake of ferric iron-containing transferrin. Ferric iron can then undergo reduction to ferrous iron by ferrireductases inside endosomes followed by DMT1-mediated pumping into the cytosol and subsequently cellular export by ferroportin. The expression of soluble ceruloplasmin by brain endothelial cells, pericytes, and astrocytes that together form the neurovascular unit (NVU) provides the ferroxidase activity necessary to reoxidize ferrous iron once released inside the brain.

Expression of Iron-Related Proteins at the Neurovascular Unit Supports Reduction and Reoxidation of Iron for Transport Through the Blood-Brain Barrier.

The functional receptor for type III interferons (IFNs) is a heterodimer of IFNLR1 and IL10R2. IFNLR1 is expressed in a highly tissue specific manner, with epithelial and liver tissue as the prime expressing tissues in humans. However, knowledge about the molecular pathways responsible for regulating the expression of IFNLR1 is yet unknown. In this study, various bioinformatics tools were used to predict the scores of signal peptides of IFNλR1 and IFNαR1, which was considered as an important difference in the expression of both receptors or participation in regulating the IFNLR1 gene. In silico study revealed that the signal peptide of IFNαR1 had more potential than the signal peptide of IFNλR1 but changing the signal peptide of wild type IFNλR1 with the signal peptide of IFNαR1 in wet lab had barely shown any differences. Selective expression of IFNλR1 was considered to be a plus point towards the targeted anti-viral activity of IFNλs but artificial control on its expression will surely make IFNλs a better drug with enhanced activity. The results of this study may help us in contributing some understanding towards the mechanisms involved in the selective expression of IFNLR1 and exceptionalities involved.

Unraveling the molecular mechanism governing the tissue specific expression of IFNλR1.

Brain capillary endothelium mediates the exchange of nutrients between blood and brain parenchyma. This barrier function of the brain capillaries also limits passage of pharmaceuticals from blood to brain, which hinders treatment of several neurological disorders. Receptor-mediated transport has been suggested as a potential pharmaceutical delivery route across the brain endothelium, e.g. reports have shown that the transferrin receptor (TfR) facilitates transcytosis of TfR antibodies, but it is not known whether this recycling receptor itself traffics from apical to basal membrane in the process. Here, we elucidate the endosomal trafficking of the retrograde transported cation-independent mannose-6-phosphate receptor (MPR300) in primary cultures of brain endothelial cells (BECs) of porcine and bovine origin. Receptor expression and localisation of MPR300 in the endo-lysosomal system and trafficking of internalised receptor are analysed. We also demonstrate that MPR300 can undergo bidirectional apical-basal trafficking in primary BECs in co-culture with astrocytes. This is, to our knowledge, the first detailed study of retrograde transported receptor trafficking in BECs, and the study demonstrates that MPR300 can be transported from the luminal to abluminal membrane and reverse. Such trafficking of MPR300 suggests that retrograde transported receptors in general may provide a mechanism for transport of pharmaceuticals into the brain.

Bidirectional apical-basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells.

Drug delivery to the brain is hampered by the presence of the blood-brain barrier, which excludes most molecules from freely diffusing into the brain, and tightly regulates the active transport mechanisms that ensure sufficient delivery of nutrients to the brain parenchyma. Harnessing the possibility of delivering neuroactive drugs by way of receptors already present on the brain endothelium has been of interest for many years. The transferrin receptor is of special interest since its expression is limited to the endothelium of the brain as opposed to peripheral endothelium. Here, we investigate the possibility of delivering immunoliposomes and their encapsulated cargo to the brain via targeting of the transferrin receptor. We find that transferrin receptor-targeting increases the association between the immunoliposomes and primary endothelial cells in vitro, but that this does not correlate with increased cargo transcytosis. Furthermore, we show that the transferrin receptor-targeted immunoliposomes accumulate along the microvessels of the brains of rats, but find no evidence for transcytosis of the immunoliposome. Conversely, the increased accumulation correlated both with increased cargo uptake in the brain endothelium and subsequent cargo transport into the brain. These findings suggest that transferrin receptor-targeting is a relevant strategy of increasing drug exposure to the brain.

Targeting transferrin receptors at the blood-brain barrier improves the uptake of immunoliposomes and subsequent cargo transport into the brain parenchyma.

Piotr Siupka

Department of Biomedicine,
Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery,
Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Department of Biomedicine

Aarhus University

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Piotr Siupka

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedicine,Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery,Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Department of Biomedicine

Aarhus University

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Department of Biomedicine,
Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery,
Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Department of Biomedicine