Establishment of a high-content imaging assay for tau aggregation in hiPSC-derived neurons differentiated from two protocols to routinely evaluate compounds and genetic perturbations.

Aberrant protein aggregation is a pathological cellular hallmark of many neurodegenerative diseases, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), where the tau protein is aggregating, forming neurofibrillary tangles (NFTs), and propagating from neuron to neuron. These processes have been linked to disease progression and a decline in cognitive function. Various therapeutic approaches aim at the prevention or reduction of tau aggregates in neurons. Human induced pluripotent stem cells (hiPSCs) are a very valuable tool in neuroscience discovery, as they offer access to potentially unlimited amounts of cell types that are affected in disease, including cortical neurons, for in vitro studies. We have generated an in vitro model for tau aggregation that uses hiPSC - derived neurons expressing an aggregation prone, fluorescently tagged version of the human tau protein after lentiviral transduction. Upon addition of tau seeds in the form of recombinant sonicated paired helical filaments (sPHFs), the neurons show robust, disease-like aggregation of the tau protein. The model was developed as a plate-based high content screening assay coupled with an image analysis algorithm to evaluate the impact of small molecules or genetic perturbations on tau. We show that the assay can be used to evaluate small molecules or screen targeted compound libraries. Using siRNA-based gene knockdown, genes of interest can be evaluated, and we could show that a targeted gene library can be screened, by screening nearly 100 deubiquitinating enzymes (DUBs) in that assay. The assay uses an imaging-based readout, a relatively short timeline, quantifies the extent of tau aggregation, and also allows the assessment of cell viability. Furthermore, it can be easily adapted to different hiPSC lines or neuronal subtypes. Taken together, this complex and highly relevant approach can be routinely applied on a weekly basis in the screening funnels of several projects and generates data with a turnaround time of approximately five weeks.

Novel Developments to Enable Treatment of CNS Diseases with Targeted Drug Delivery.

The blood-brain barrier (BBB) is a major hurdle for the development of systemically delivered drugs against diseases of the central nervous system (CNS). Because of this barrier there is still a huge unmet need for the treatment of these diseases, despite years of research efforts across the pharmaceutical industry. Novel therapeutic entities, such as gene therapy and degradomers, have become increasingly popular in recent years, but have not been the focus for CNS indications so far. To unfold their full potential for the treatment of CNS diseases, these therapeutic entities will most likely have to rely on innovative delivery technologies. Here we will describe and assess approaches, both invasive and non-invasive, that can enable, or at least increase, the probability of a successful drug development of such novel therapeutics for CNS indications.

Robust LC3B lipidation analysis by precisely adjusting autophagic flux.

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.

Trafficking of JC virus-like particles across the blood-brain barrier.

Hollow viral vectors, such as John Cunningham virus-like particles (JC VLPs), provide a unique opportunity to deliver drug cargo into targeted cells and tissue. Current understanding of the entry of JC virus in brain cells has remained insufficient. In particular, interaction of JC VLPs with the blood-brain barrier (BBB) has not been analyzed in detail. Thus, JC VLPs were produced in this study for investigating the trafficking across the BBB. We performed a carotid artery injection procedure for mouse brain to qualitatively study JC VLPs' in vivo binding and distribution and used in vitro approaches to analyze their uptake and export kinetics in brain endothelial cells. Our results show that clathrin-dependent mechanisms contributed to the entry of VLPs into brain endothelial cells, and exocytosis or transcytosis of VLPs across the BBB was observed in vitro. VLPs were found to interact with sialic acid glycans in mouse brain endothelia. The ability of JC VLPs to cross the BBB can be useful in developing a delivery system for transport of genes and small molecule cargoes to the brain.

Electronic Lab Notebooks and Experimental Design Assistants.

Documentation of experiments is essential for best research practice and ensures scientific transparency and data integrity. Traditionally, the paper lab notebook (pLN) has been employed for documentation of experimental procedures, but over the course of the last decades, the introduction of electronic tools has changed the research landscape and the way that work is performed. Nowadays, almost all data acquisition, analysis, presentation and archiving are done with electronic tools. The use of electronic tools provides many new possibilities, as well as challenges, particularly with respect to documentation and data quality. One of the biggest hurdles is the management of data on different devices with a substantial amount of metadata. Transparency and integrity have to be ensured and must be reflected in documentation within LNs. With this in mind, electronic LNs (eLN) were introduced to make documentation of experiments more straightforward, with the development of enhanced functionality leading gradually to their more widespread use. This chapter gives a general overview of eLNs in the scientific environment with a focus on the advantages of supporting quality and transparency of the research. It provides guidance on adopting an eLN and gives an example on how to set up unique Study-IDs in labs in order to maintain and enhance best practices. Overall, the chapter highlights the central role of eLNs in supporting the documentation and reproducibility of experiments.

Brain delivery research in public-private partnerships: The IMI-JU COMPACT consortium as an example.

The Blood-Brain Barrier (BBB) represents a major hurdle in the development of treatments for CNS disorders due to the fact that it very effectively keeps drugs, especially biological macromolecules, out of the brain. Concomitantly with the increasing importance of biologics research on the BBB and, more specifically, on brain delivery technologies has intensified in recent years. Public-Private Partnerships (PPPs) represent an innovative opportunity to address such complex challenges as they bring together the best expertise from both industry and academia. Here we present the IMI-JU COMPACT (Collaboration on the Optimisation of Macromolecular Pharmaceutical Access to Cellular Targets) consortium working on nanocarriers for targeted delivery of macromolecules as an example. The scope of the consortium, its goals and the expertise within the consortium are outlined. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders".

Absence of Foxp3+ regulatory T cells during allergen provocation does not exacerbate murine allergic airway inflammation.

Regulatory T cells (Tregs) play a non-redundant role in maintenance of immune homeostasis. This is achieved by suppressing both, priming of naïve cells and effector cell functions. Although Tregs have been implicated in modulating allergic immune responses, their influence on distinct phases of development of allergies remains unclear. In this study, by using bacterial artificial chromosome (BAC)-transgenic Foxp3-DTR (DEREG) mice we demonstrate that the absence of Foxp3(+) Tregs during the allergen challenge surprisingly does not exacerbate allergic airway inflammation in BALB/c mice. As genetic disposition due to strain specificity may contribute significantly to development of allergies, we performed similar experiment in C57BL/6 mice, which are less susceptible to allergy in the model of sensitization used in this study. We report that the genetic background does not influence the consequence of this depletion regimen. These results signify the temporal regulation exerted by Foxp3(+) Tregs in limiting allergic airway inflammation and may influence their application as potential therapeutics.

Optimal isolation of functional Foxp3+ induced regulatory T cells using DEREG mice.

Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells) mice have greatly helped in exploring the biology of Foxp3(+) Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC)-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3(+) Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs) are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3(+) iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4(+)Foxp3(+) iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP(+)Foxp3(-) cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP(-) cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP(+)Foxp3(+) iTregs exhibited potent suppressive activity comparable to that of natural eGFP(+)Foxp3(+) Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP(+)Foxp3(+) iTregs by use of DEREG mice. Isolation of functional Foxp3(+) iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP expression are driven from distinct Foxp3 loci and because this dichotomy preferentially occurs only under defined in vitro conditions.

Fast investigations from biological matrices using CE--test of a blood-brain barrier model.

In order to adopt a general workflow for complex biological matrices with respect to a new blood-brain barrier (BBB) model, a micellar electrokinetic chromatography method has been developed. The cells forming the BBB have been cultivated in a special cell growth medium in which six drugs (acetaminophen, caffeine, carbamazepine, cimetidine, indometacin and propranolol) have been dissolved and tested for their penetration properties. The results showed good to very good accordance to the reference values. Samples were directly injected onto the capillary without any pretreatment (fused silica capillary, id: 50 µm, L: 48 cm, l: 40 cm). After method development, separations were carried out using a 60 mM borate buffer containing 200 mM of SDS at 30 kV, leading to an analysis time of less than 10 min. Between two runs the capillary was rinsed with a mixture of equal parts of running buffer and isopropanol (70% v/v), which proved to be very effective to remove matrix compounds. An appropriate choice of the detection wavelength (220 or 254 nm) could avoid major interferences between analytes and matrix. The typical RSD% for migration times was approximately 2%, for peak areas, it ranged from 2 to 6%, which was very well acceptable for the generic method used in this study and the low concentrations investigated. The LODs ranged from 10 to 30 ng/mL.

An optimized in vitro blood-brain barrier model reveals bidirectional transmigration of African trypanosome strains.

The transmigration of African trypanosomes across the human blood-brain barrier (BBB) is the critical step during the course of human African trypanosomiasis. The parasites Trypanosoma brucei gambiense and T. b. rhodesiense are transmitted to humans during the bite of tsetse flies. Trypanosomes multiply within the bloodstream and finally invade the central nervous system (CNS), which leads to the death of untreated patients. This project focused on the mechanisms of trypanosomal traversal across the BBB. In order to establish a suitable in vitro BBB model for parasite transmigration, different human cell lines were used, including ECV304, HBMEC and HUVEC, as well as C6 rat astrocytes. Validation of the BBB models with Escherichia coli HB101 and E. coli K1 revealed that a combination of ECV304 cells seeded on Matrigel as a semi-synthetic basement membrane and C6 astrocytes resulted in an optimal BBB model system. The BBB model showed selective permeability for the pathogenic E. coli K1 strain, and African trypanosomes were able to traverse the optimized ECV304-C6 BBB efficiently. Furthermore, coincubation indicated that paracellular macrophage transmigration does not facilitate trypanosomal BBB traversal. An inverse assembly of the BBB model demonstrated that trypanosomes were also able to transmigrate the optimized ECV304-C6 BBB backwards, indicating the relevance of the CNS as a possible reservoir of a relapsing parasitaemia.