Innate immunity in the Grid2Lc/+ mouse model of cerebellar neurodegeneration: glial CD95/CD95L plays a non-apoptotic role in persistent neuron loss-associated inflammatory reactions in the cerebellum.

BACKGROUND: There is growing evidence that the death receptor CD95 has a wider role in non-apoptotic functions. In the brain, it may contribute to neural death and to the associated inflammatory reaction via a non-apoptotic pathway. Brain injury triggers an inflammatory reaction in which the CD95/CD95L system acts principally through peripheral cells recruited to the lesion. In cases of inflammation within the brain, with no blood-brain barrier leakage, the role of the CD95/CD95L system is thus unclear. We investigated the possible role of CD95 and CD95L in such conditions, by studying the relationships between glial cell activation, neuron death and CD95/CD95L expression in the cerebellum of the Lurcher (Grid2(Lc/+)) mutant mouse, a model of cerebellar neurodegeneration. METHODS: Glial cells in slices of wild-type and Lurcher mouse cerebella were observed by light microscopy at various ages overlapping periods of neuron loss and of pre- and post-neurodegeneration. Subcellular organization was studied by electron microscopy. We assessed CD95 levels by western blotting, RT-PCR and glial cell cultures. The levels of CD95L and IL-6 were studied by ELISA and a biological assay, respectively. RESULTS: In the Grid2(Lc/+)cerebellum, neuron loss triggers a typical, but abnormally persistent, inflammatory reaction. We identified two phases of astrogliosis: an early burst of large glial cell activation, peaking at postnatal days 25 to 26, coinciding with peak cerebellar neuron loss, followed by a long period of slow decline indicating that the strength of the glial reaction is modulated by neuron mortality rates. Comparisons of time-courses of glial cell activation, cytokine production and neuron loss revealed that the number of surviving neurons decreased as CD95 increased. Thus, CD95 cannot be directly involved in neuron death, and its role must be limited to a contribution to the inflammatory reaction. The upregulation of CD95 likely on astrocytes coincides with increases in the levels of IL-6, a cytokine produced principally by astrocytes, and soluble CD95L. CONCLUSIONS: These results suggest that CD95 and soluble CD95L contribute, via non-apoptotic signaling, to the inflammatory reaction initiated early in neuron death within the Grid2(Lc/+) cerebellum.

RORalpha, a key to the development and functioning of the brain.

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.

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

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.

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

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.

A new and simple approach for genotyping Alzheimer's disease presenilin-1 mutant knock-in mice.

The use of transgenic mice expressing point mutations demands that the detection of the different alleles is efficient and reliable. In addition, the multiplication of transgenes included in mouse models of human disease underlines the importance of correct controls and the fact that investigators need an accurate and rapid genotyping of the littermates generated. In this study, we demonstrate a powerful alternative for genotyping using presenilin-1 mutant knock-in (PS1M146KI) mice as an example. Mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer's disease (AD). PS1M146VKI mice that express the PS1M146V targeted allele at normal physiological levels and triple-transgenic model (3 x Tg-AD) derived from homozygous PS1M146VKI mice were generated to study the pathogenesis of AD. Genotyping PS1M146VKI line requires many steps and thus a large quantity of DNA. In PS1M146VKI mice, only three nucleotides are modified in the gene. Here we show that this small mutated DNA sequence can affect its secondary structure resulting in altered mobility that can be easily detected on a polyacrylamide gel, by the single-strand conformation polymorphism (SSCP) technique. Our results demonstrate that SSCP is a simple, accurate, repeatable and efficient method for the routine genotyping of this current AD model. This method could be easily applied to other transgenic mice.

Quantitative analysis of microglial cells in the degenerating cerebellum of the staggerer (RORA(sg/sg)) mutant mouse.

Elevated levels of pro-inflammatory cytokines, such as IL-1ss and IL-6, have been detected in the cerebellum of Rora(sg/sg) mice during the first postnatal month of neurodegenerative process. This suggests the existence of a microglial reaction in the context of an inflammatory process that would be triggered by the massive neuronal loss. To test this hypothesis, we qualitatively and quantitatively studied the microglial cell population using lectin and nucleosidic diphosphatase labeling of the cerebellum of 30-day-old mice. The massive neuronal loss induces a 11.7-fold smaller size of the Rora(sg/sg) cerebellum compared to wild-types. We showed that the Rora(sg/sg) microglia population is exclusively composed of cells displaying the characteristic morphology of activated cells, with enlarged, heavily stained cell bodies and few thick processes, in contrast to microglial cells in the wild-type. The density of microglia is 2.7-fold higher in Rora(sg/sg) than wild-type mice (22444+/-5011 cells/mm(3) versus 8158+/-1584 cells/mm(3)), although the absolute number is 4-fold smaller. These results show that neurodegeneration in the Rora(sg/sg) cerebellum leads to persistance of microglial activation while in wild-type it disappears around P10.