Search

Background The pathways that control protein transport across the blood–brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. Methods We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. Results BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. Conclusions Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Targeting receptor-mediated transcytosis (RMT) is a successful strategy for drug delivery of biologic agents across the blood-brain barrier (BBB). The recent development of human BBB organoid models is a major advancement to help characterize the mechanisms of RMT and thus accelerate the design of brain delivery technologies. BBB organoids exhibit self-organization, which resembles the architecture of the neurovascular unit, and low paracellular permeability, due to the formation of tight junctions between endothelial cells. However, current methods of organoid generation have low throughput, exhibit substantial heterogeneity across experiments, and require extensive manual handling. These limitations prevent the use of BBB organoids as a screening tool for discovery and optimization of therapeutic molecules. In this protocol, we use hydrogel-based arrays to generate human BBB organoids, with a 35-fold increase in organoid yield as compared to previous protocols using 96-well plates. We incubate BBB organoid arrays with monoclonal antibody-based constructs and use a custom semi-automated imaging assay to assess RMT within the organoid core. The experimental and analytical tools described in this protocol provide a scalable platform that can be incorporated in the early stages of drug discovery to accelerate the development and optimization of brain delivery technologies to cross the BBB.

Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In particular, the inner blood-retinal barrier (iBRB) and blood–brain barrier (BBB) share common origins in development, and similar morphology and function in adult tissue, while barrier breakdown and leakage of neurotoxic molecules can be accompanied by neurodegeneration. Therefore, pre-clinical research requires human in vitro models that elucidate pathophysiological mechanisms and support drug discovery, to add to animal in vivo modeling that poorly predict patient responses. Advanced cellular models such as microphysiological systems (MPS) recapitulate tissue organization and function in many organ-specific contexts, providing physiological relevance, potential for customization to different population groups, and scalability for drug screening purposes. While human-based MPS have been developed for tissues such as lung, gut, brain and tumors, few comprehensive models exist for ocular tissues and iBRB modeling. Recent BBB in vitro models using human cells of the neurovascular unit (NVU) showed physiological morphology and permeability values, and reproduced brain neurological disorder phenotypes that could be applicable to modeling the iBRB. Here, we describe similarities between iBRB and BBB properties, compare existing neurovascular barrier models, propose leverage of MPS-based strategies to develop new iBRB models, and explore potentials to personalize cellular inputs and improve pre-clinical testing.

For therapeutic proteins, the currently established standard development path generally does not foresee biotransformation studies by default because it is well known that the clearance of therapeutic proteins proceeds via degradation to small peptides and individual amino acids. In contrast to small molecules, there is no general need to identify enzymes involved in biotransformation because this information is not relevant for drug–drug interaction assessment and for understanding the clearance of a therapeutic protein. Nevertheless, there are good reasons to embark on biotransformation studies, especially for complex therapeutic proteins. Typical triggers are unexpected rapid clearance, species differences in clearance not following the typical allometric relationship, a mismatch in the pharmacokinetics/pharmacodynamics (PK/PD) relationship, and the need to understand observed differences between the results of multiple bioanalytical methods (e.g., total vs. target-binding competent antibody concentrations). Early on during compound optimization, knowledge on protein biotransformation may help to design more stable drug candidates with favorable in vivo PK properties. Understanding the biotransformation of a therapeutic protein may also support designing and understanding the bioanalytical assay and ultimately the PK/PD assessment. Especially in cases where biotransformation products are pharmacologically active, quantification and assessment of their contribution to the overall pharmacological effect can be important for establishing a PK/PD relationship and extrapolation to humans. With the increasing number of complex therapeutic protein formats, the need for understanding the biotransformation of therapeutic proteins becomes more urgent. This article provides an overview on biotransformation processes, proteases involved, strategic considerations, regulatory guidelines, literature examples for in vitro and in vivo biotransformation, and technical approaches to study protein biotransformation. SIGNIFICANCE STATEMENT Understanding the biotransformation of complex therapeutic proteins can be crucial for establishing a pharmacokinetic/pharmacodynamic relationship. This article will highlight scientific, strategic, regulatory, and technological features of protein biotransformation.

BackgroundThe pathways that control protein transport across the Blood-Brain Barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development.MethodsWe applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis.ResultsBBB organoid arrays allowed the simultaneous growth of more than 5000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis.ConclusionsHuman BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood–brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood–brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19., A novel study led by scientists in Lübeck, Germany, shows that SARS-CoV-2-infected brain endothelial cells undergo cell death due to the cleavage of NEMO by the viral protease Mpro, potentially causing cerebral COVID-19 and ‘long COVID’ symptoms.

The blood-brain barrier is a dynamic multicellular interface that regulates the transport of molecules between the blood circulation and the brain parenchyma. Proteins and peptides required for brain homeostasis cross the blood-brain barrier via transcellular transport, but the mechanisms that control this pathway are not well characterized. Here, we highlight recent studies on intracellular transport and transcytosis across the blood-brain barrier. Endothelial cells at the blood-brain barrier possess an intricate endosomal network that allows sorting to diverse cellular destinations. Internalization from the plasma membrane, endosomal sorting, and exocytosis all contribute to the regulation of transcytosis. Transmembrane receptors and blood-borne proteins utilize different pathways and mechanisms for transport across brain endothelial cells. Alterations to intracellular transport in brain endothelial cells during diseases of the central nervous system contribute to blood-brain barrier disruption and disease progression. Harnessing the intracellular sorting mechanisms at the blood-brain barrier can help improve delivery of biotherapeutics to the brain.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

The blood–brain barrier (BBB) regulates differing needs of the various brain regions by controlling transport of blood-borne components from the neurovascular circulation into the brain parenchyma. The mechanisms underlying region-specific transport across the BBB are not completely understood. Previous work showed that pericytes are key regulators of BBB function. Here we investigated whether pericytes influence BBB permeability in a region-specific manner by analysing the regional permeability of the BBB in the pdgf-b ret/ret mouse model of pericyte depletion. We show that BBB permeability is heterogeneous in pdgf-b ret/ret mice, being significantly higher in the cortex, striatum and hippocampus compared to the interbrain and midbrain. However, we show that this regional heterogeneity in BBB permeability is not explained by local differences in pericyte coverage. Region-specific differences in permeability were not associated with disruption of tight junctions but may result from changes in transcytosis across brain endothelial cells. Our data show that certain brain regions are able to maintain low BBB permeability despite substantial pericyte loss and suggest that additional, locally-acting mechanisms may contribute to control of transport.

Summary Transcytosis across the blood-brain barrier (BBB) regulates key processes of the brain, but the intracellular sorting mechanisms that determine successful receptor-mediated transcytosis in brain endothelial cells (BECs) remain unidentified. Here, we used Transferrin receptor-based Brain Shuttle constructs to investigate intracellular transport in BECs, and we uncovered a pathway for the regulation of receptor-mediated transcytosis. By combining live-cell imaging and mathematical modeling in vitro with super-resolution microscopy of the BBB, we show that intracellular tubules promote transcytosis across the BBB. A monovalent construct (sFab) sorted for transcytosis was localized to intracellular tubules, whereas a bivalent construct (dFab) sorted for degradation formed clusters with impaired transport along tubules. Manipulating tubule biogenesis by overexpressing the small GTPase Rab17 increased dFab transport into tubules and induced its transcytosis in BECs. We propose that sorting tubules regulate transcytosis in BECs and may be a general mechanism for receptor-mediated transport across the BBB.

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-bret/ret mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain.

SummaryBackgroundEndocytosis allows the import and distribution of cargo into a series of endosomes with distinct morphological and biochemical characteristics. Our current understanding of endocytic cargo trafficking is based on the kinetics of net cargo transport between endosomal compartments without considering individual endosomes. However, endosomes form a dynamic network of membranes undergoing fusion and fission, thereby continuously exchanging and redistributing cargo. The macroscopic kinetic properties, i.e., the properties of the endosomal network as a whole, result from the collective behaviors of many individual endosomes, a problem so far largely unaddressed.ResultsHere, we developed a general theoretical framework to describe the dynamics of cargo distributions in the endosomal network. We combined the theory with quantitative experiments to study how the macroscopic kinetic properties of the endosomal network emerge from microscopic processes at the level of individual endosomes. We compared our theory predictions to experimental data in which dynamic distributions of endocytosed low-density lipoprotein (LDL) were quantified.ConclusionsOur theory can quantitatively describe the observed cargo distributions as a function of time. Remarkably, the theory allows determining microscopic kinetic parameters such as the fusion rate between endosomes from still images of cargo distributions at different times of internalization. We show that this method is robust and sensitive because cargo distributions result from an average over many stochastic events in many cells. Our results provide theoretical and experimental support to the “funnel model” of endosome progression and suggest that the conversion of early to late endosomes is the major mode of LDL trafficking.

Fibroblast transformation by H-RasG12V induces internalization of PDGFRβ by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. It is proposed that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling through increased receptor macropinocytosis., Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.

The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. In this work we analyzed the response of two different insect orders, the Lepidopteran Manduca sexta and Dipteran Aedes aegypti to highly specific Cry toxins, Cry1Ab and Cry11Aa, respectively. One pathway activated in different organisms in response to a variety of pore-forming toxins is the mitogen-activated protein kinase p38 pathway (MAPK p38) that activates a complex defense response. We analyzed the MAPK p38 activation by immunodetection of its phosphorylated isoform, and the induction of p38 by RT-PCR, real-time PCR quantitative assays and immunodetection. We show that MAPK p38 is activated at postraductional level after Cry toxin intoxication in both insect orders. We detected the p38 induction at the transcriptional and traductional level, and observed a different response. In these three levels, we found that both insects respond to Cry toxin action but M. sexta responses more strongly than A. aegypti. Gene silencing of MAPK p38 in vivo, resulted in both insect species becoming hypersensitive to Cry toxin action, suggesting that the MAPK p38 pathway is involved in insect defense against Bt Cry toxins. This finding may have biotechnological applications for enhancing the activity of some Bt Cry toxins against specific insect pests.

Supercharged proteins can deliver functional macromolecules into the cytoplasm of mammalian cells with potencies that exceed those of cationic peptides. The structural features of supercharged proteins that determine their delivery effectiveness and the intracellular fate of supercharged proteins once they enter cells have not yet been studied. Using a large set of supercharged GFP (scGFP) variants, we found that the level of cellular uptake is sigmoidally related to net charge, and that scGFPs enter cells through multiple pathways including clathrin-dependent endocytosis and macropinocytosis. Supercharged proteins activate Rho and ERK1/2, and also alter the endocytic transport of transferrin and EGF. Finally, we discovered that the intracellular trafficking of endosomes containing scGFPs is altered in a manner that correlates with protein delivery potency. Collectively, our findings establish basic structure-activity relationships of supercharged proteins and implicate the modulation of endosomal trafficking as a determinant of cell-penetration and macromolecule-delivery efficiency.

Cells need to decode chemical or physical signals from their environment in order to make decisions on their fate. In the case of signalling receptors, ligand binding triggers a cascade of chemical reactions but also the internalization of the activated receptors in the endocytic pathway. Here, we highlight recent studies revealing a new role of the endosomal network in signal processing. The diversity of entry pathways and endosomal compartments is exploited to regulate the kinetics of receptor trafficking, and interactions with specific signalling adaptors and effectors. By governing the spatio-temporal distribution of signalling molecules, the endosomal system functions analogously to a digital-analogue computer that regulates the specificity and robustness of the signalling response.