HIF prolyl hydroxylase 2/3 deletion disrupts astrocytic integrity and exacerbates neuroinflammation.

Astrocytes constitute the parenchymal border of the blood-brain barrier (BBB), modulate the exchange of soluble and cellular elements, and are essential for neuronal metabolic support. Thus, astrocytes critically influence neuronal network integrity. In hypoxia, astrocytes upregulate a transcriptional program that has been shown to boost neuroprotection in several models of neurological diseases. We investigated transgenic mice with astrocyte-specific activation of the hypoxia-response program by deleting the oxygen sensors, HIF prolyl-hydroxylase domains 2 and 3 (Phd2/3). We induced astrocytic Phd2/3 deletion after onset of clinical signs in experimental autoimmune encephalomyelitis (EAE) that led to an exacerbation of the disease mediated by massive immune cell infiltration. We found that Phd2/3-ko astrocytes, though expressing a neuroprotective signature, exhibited a gradual loss of gap-junctional Connexin-43 (Cx43), which was induced by vascular endothelial growth factor-alpha (Vegf-a) expression. These results provide mechanistic insights into astrocyte biology, their critical role in hypoxic states, and in chronic inflammatory CNS diseases.

Recellularized Native Kidney Scaffolds as a Novel Tool in Nephrotoxicity Screening.

Drug-induced kidney injury in medicinal compound development accounts for over 20% of clinical trial failures and involves damage to different nephron segments, mostly the proximal tubule. Yet, currently applied cell models fail to reliably predict nephrotoxicity; neither are such models easy to establish. Here, we developed a novel three-dimensional (3D) nephrotoxicity platform on the basis of decellularized rat kidney scaffolds (DS) recellularized with conditionally immortalized human renal proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1). A 5-day SDS-based decellularization protocol was used to generate DS, of which 100-µm slices were cut and used for cell seeding. After 8 days of culturing, recellularized scaffolds (RS) demonstrated 3D-tubule formation along with tubular epithelial characteristics, including drug transporter function. Exposure of RS to cisplatin (CDDP), tenofovir (TFV), or cyclosporin A (CsA) as prototypical nephrotoxic drugs revealed concentration-dependent reduction in cell viability, as assessed by PrestoBlue and Live/Dead staining assays. This was most probably attributable to specific uptake of CDDP by the organic cation transporter 2 (OCT2), TFV through organic anion transporter 1 (OAT1), and CsA competing for P-glycoprotein-mediated efflux. Compared with 2D cultures, RS showed an increased sensitivity to cisplatin and tenofovir toxicity after 24-hour exposure (9 and 2.2 fold, respectively). In conclusion, we developed a physiologically relevant 3D nephrotoxicity screening platform that could be a novel tool in drug development.

Inflammatory Cytokines Associated with Multiple Sclerosis Directly Induce Alterations of Neuronal Cytoarchitecture in Human Neurons.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) coined by inflammation and neurodegeneration. The actual cause of the neurodegenerative component of the disease is however unclear. We investigated here the direct and differential effects of inflammatory mediators on human neurons. We used embryonic stem cell-derived (H9) human neuronal stem cells (hNSC) to generate neuronal cultures. Neurons were subsequently treated with tumour necrosis factor alpha (TNFα), interferon gamma (IFNγ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 17A (IL-17A) and interleukin 10 (IL-10) separately or in combination. Immunofluorescence staining and quantitative polymerase chain reaction (qPCR) were used to assess cytokine receptor expression, cell integrity and transcriptomic changes upon treatment. H9-hNSC-derived neurons expressed cytokine receptors for IFNγ, TNFα, IL-10 and IL-17A. Neuronal exposure to these cytokines resulted in differential effects on neurite integrity parameters with a clear decrease for TNFα- and GM-CSF-treated neurons. The combinatorial treatment with IL-17A/IFNγ or IL-17A/TNFα induced a more pronounced effect on neurite integrity. Furthermore, combinatorial treatments with two cytokines induced several key signalling pathways, i.e. NFκB-, hedgehog and oxidative stress signalling, stronger than any of the cytokines alone. This work supports the idea of immune-neuronal crosstalk and the need to focus on the potential role of inflammatory cytokines on neuronal cytoarchitecture and function.

iPSC-derived reactive astrocytes from patients with multiple sclerosis protect cocultured neurons in inflammatory conditions.

Multiple sclerosis (MS) is the most common chronic central nervous system inflammatory disease. Individual courses are highly variable, with complete remission in some patients and relentless progression in others. We generated induced pluripotent stem cells (iPSCs) to investigate possible mechanisms in benign MS (BMS), compared with progressive MS (PMS). We differentiated neurons and astrocytes that were then stressed with inflammatory cytokines typically associated with MS phenotypes. TNF-α/IL-17A treatment increased neurite damage in MS neurons from both clinical phenotypes. In contrast, TNF-α/IL-17A-reactive BMS astrocytes cultured with healthy control neurons exhibited less axonal damage compared with PMS astrocytes. Accordingly, single-cell transcriptomic BMS astrocyte analysis of cocultured neurons revealed upregulated neuronal resilience pathways; these astrocytes showed differential growth factor expression. Furthermore, supernatants from BMS astrocyte/neuronal cocultures rescued TNF-α/IL-17-induced neurite damage. This process was associated with a unique LIF and TGF-ß1 growth factor expression, as induced by TNF-α/IL-17 and JAK-STAT activation. Our findings highlight a potential therapeutic role of modulation of astrocyte phenotypes, generating a neuroprotective milieu. Such effects could prevent permanent neuronal damage.

Identification of the gliogenic state of human neural stem cells to optimize in vitro astrocyte differentiation.

BACKGROUND: Human preclinical models are crucial for advancing biomedical research. In particular consistent and robust protocols for astrocyte differentiation in the human system are rare. NEW METHOD: We performed a transcriptional characterization of human gliogenesis using embryonic H9- derived hNSCs. Based on these findings we established a fast and highly efficient protocol for the differentiation of mature human astrocytes. We could reproduce these results in induced pluripotent stem cell (iPSC)-derived NSCs. RESULTS: We identified an increasing propensity of NSCs to give rise to astrocytes with repeated cell passaging. The gliogenic phenotype of NSCs was marked by a down-regulation of stem cell factors (e.g. SOX1, SOX2, EGFR) and an increase of glia-associated factors (e.g. NFIX, SOX9, PDGFRa). Using late passage NSCs, rapid and robust astrocyte differentiation can be achieved within 28 days. COMPARISON WITH EXISTING METHOD(S): In published protocols it usually takes around three months to yield in mature astrocytes. The difficulty, expense and time associated with generating astrocytes in vitro represents a major roadblock for glial cell research. We show that rapid and robust astrocyte differentiation can be achieved within 28 days. We describe here by an extensive sequential transcriptome analysis of hNSCs the characterization of the signature of a novel gliogenic stem cell population. The transcriptomic signature might serve to identify the proper divisional maturity. CONCLUSIONS: This work sheds light on the factors associated with rapid NSC differentiation into glial cells. These findings contribute to understand human gliogenesis and to develop novel preclinical models that will help to study CNS disease such as Multiple Sclerosis.

Pentafluorosulfanyl (SF5) as a Superior 19F Magnetic Resonance Reporter Group: Signal Detection and Biological Activity of Teriflunomide Derivatives.

Fluorine (19F) magnetic resonance imaging (MRI) is severely limited by a low signal-to noise ratio (SNR), and tapping it for 19F drug detection in vivo still poses a significant challenge. However, it bears the potential for label-free theranostic imaging. Recently, we detected the fluorinated dihydroorotate dehydrogenase (DHODH) inhibitor teriflunomide (TF) noninvasively in an animal model of multiple sclerosis (MS) using 19F MR spectroscopy (MRS). In the present study, we probed distinct modifications to the CF3 group of TF to improve its SNR. This revealed SF5 as a superior alternative to the CF3 group. The value of the SF5 bioisostere as a 19F MRI reporter group within a biological or pharmacological context is by far underexplored. Here, we compared the biological and pharmacological activities of different TF derivatives and their 19F MR properties (chemical shift and relaxation times). The 19F MR SNR efficiency of three MRI methods revealed that SF5-substituted TF has the highest 19F MR SNR efficiency in combination with an ultrashort echo-time (UTE) MRI method. Chemical modifications did not reduce pharmacological or biological activity as shown in the in vitro dihydroorotate dehydrogenase enzyme and T cell proliferation assays. Instead, SF5-substituted TF showed an improved capacity to inhibit T cell proliferation, indicating better anti-inflammatory activity and its suitability as a viable bioisostere in this context. This study proposes SF5 as a novel superior 19F MR reporter group for the MS drug teriflunomide.

Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes.

BACKGROUND: Molecular analyses of cell populations and single cells have been instrumental in the advancement of our understanding of the physiology and pathologic processes of the nervous system. However, the limitation of these methods is the dependence on a gentle, efficient and specific enrichment procedure for the target cell population. In particular, this has been challenging for tightly interconnected cells, for example central nervous system (CNS) endogenous cells such as astrocytes. NEW METHOD: Here we adopted one of the most common methods of cell extraction, namely, enzymatic tissue digestion followed by fluorescence-activated cell sorting (FACS) of individual cells. We evaluated different enzymatic/mechanical tissue dissociation procedures and analyzed different astrocyte lineage transgenic models. Furthermore, we compared the cell extraction efficiency from spinal cord vs. brain. RESULTS: Enzymatic digestion of CNS tissue of Glast-CreERT2x tdTomatofl/fl or Aldh1l1-CreERT2x tdTomatofl/fl followed by FACS resulted in highly purified astrocytes. Automated tissue digestion strongly improved the isolated cell numbers. Aldh1l1-CreERT2 identified more astrocytes than Glast-CreERT2; isolation from brain yields higher numbers than from spinal cord. COMPARISON WITH EXISTING METHODS: We compared the efficiency and purity of the enzymatic dissociation/FACS approach with a more modern procedure consisting of tissue homogenization followed by translating ribosome affinity purification (TRAP). CONCLUSION: We found that both methods result in highly enriched astrocytic RNA. However, only TRAP isolation resulted in reliably detectable RNA concentrations from spinal cord tissue on a single animal level. Depending on the aim of the study both methods have advantages and disadvantages but both are acceptable for astrocytic RNA analysis.