combining-double-fluorescence-situ-hybridization-with-immunolabelling

Combining Double Fluorescence <em>In Situ</em> Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

University College London

Astrocytes and one of their products, IL-6, not only support neurons but also mediate inflammation in the brain. Retinoid-related orphan receptor-alpha (RORalpha) transcription factor has related roles, being neuro-protective and, in peripheral tissues, anti-inflammatory. We examined the relation of ROR(alpha) to astrocytes and IL-6 using normal and ROR(alpha) loss-of-function mutant mice. We have shown ROR(alpha) expression in astrocytes and its up-regulation by pro-inflammatory cytokines. We have also demonstrated that ROR(alpha) directly trans-activates the Il-6 gene. We suggest that this direct control is necessary to maintain IL-6 basal level in the brain and may be a link between the neuro-supportive roles of ROR(alpha), IL-6, and astrocytes. Furthermore, after inflammatory stimulation, the absence of ROR(alpha) results in excessive IL-6 up-regulation, indicating that ROR(alpha) exerts an indirect repression probably via the inhibition of the NF-kappaB signaling. Thus, our findings indicate that ROR(alpha) is a pluripotent molecular player in constitutive and adaptive astrocyte physiology.

The nuclear receptor ROR(alpha) exerts a bi-directional regulation of IL-6 in resting and reactive astrocytes.

There is increasing evidence to suggest that the neuronal response to hypoxia is regulated through their interactions with astrocytes. However, the hypoxia-induced molecular mechanisms within astrocytes which influence neuronal death have yet to be characterized. In this study, we investigated the roles of the nuclear receptor RORα (retinoid-related orphan receptor-α) respectively in neurons and astrocytes during hypoxia using cultures and cocultures of neurons and astrocytes obtained from RORα-deficient mice. We found that loss of RORα function in neuronal cultures increases neuronal death after hypoxia, suggesting a cell-autonomous neuroprotective effect of RORα. Moreover, wild-type neurons cocultured with RORα-deficient astrocytes are characterized by a higher death rate after hypoxia than neurons cocultured with wild-type astrocytes, suggesting that RORα also has a non-cell-autonomous action. By using cocultures of neurons and astrocytes of different genotypes, we showed that this neuroprotective effect of RORα in astrocytes is additive to its effect in neurons, and is mediated in part by cell-to-cell interactions between neurons and astrocytes. We also found that RORα is upregulated by hypoxia in both neurons and astrocytes. Furthermore, our data showed that RORα does not alter oxidative mechanisms during hypoxia but regulates hypoxic inducible factor 1α (HIF-1α) expression, a major regulator of hypoxia sensing, in a cell-specific manner. Indeed, the neuroprotective function of RORα in astrocytes correlates with a downregulation of HIF-1α selectively in these cells. Altogether, our results show that RORα is a key molecular player in hypoxia, protecting neurons through its dual action in neurons and astrocytes.

Cell-autonomous and non-cell-autonomous neuroprotective functions of RORα in neurons and astrocytes during hypoxia.

Studies of staggerer mice, in which retinoid-related orphan receptor-alpha (RORα) is mutated, have provided new insights into the critical functions of RORα in various physiological processes in peripheral tissues and in the brain. Staggerer mice present an ataxic phenotype caused by a massive neurodegeneration in the cerebellum. As a result, most of studies have focused on the role of RORα in the development of the cerebellum. Recent studies have expanded the role of RORα to other structures and functions in the brain. RORα was considered to be exclusively expressed in neurons in the brain. Recently, it has been shown that, in addition to its neuronal expression, RORα is expressed in glial cells and particularly in astrocytes in different brain regions. Moreover, RORα has been implicated in the regulation of some astrocyte functions such as the inflammatory function. Several reports have also presented evidence for a role of RORα in diverse pathological processes including oxidative stress-induced apoptosis and cerebral hypoxia. This review therefore focuses on the emerging roles of RORα in the brain and particularly in astrocytes.

Article	Year
The nuclear receptor ROR(alpha) exerts a bi-directional regulation of IL-6 in resting and reactive astrocytes. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19955433	2009
Cell-autonomous and non-cell-autonomous neuroprotective functions of RORα in neurons and astrocytes during hypoxia. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 21976517	2011
RORalpha, a key to the development and functioning of the brain. Cerebellum (London, England)\| PubMed ID: 22223133	2012

Sarah Jolly

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