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.

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.

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.