Vernet-der Garabedian et al. Journal of Neuroinflammation 2013, 10:65 JOURNAL OF http://www.jneuroinflammation.com/content/10/1/65 NEUROINFLAMMATION RESEARCH Open Access 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 Béatrice Vernet-der Garabedian1,2*, Paul Derer1,2, Yannick Bailly4 and Jean Mariani1,2,3 Abstract 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 (Grid2Lc/+) 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 Grid2Lc/+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 Grid2Lc/+ cerebellum. Keywords: Glial reaction, Astrocyte, Microglia, Cytokines, GFAP, IL-6, Soluble CD95L, Lurcher, Grid2 gene * Correspondence: [email protected] 1Université Pierre et Marie Curie-P6, UMR 7102, Paris F75005, France 2CNRS UMR7102, Paris F75005, France Full list of author information is available at the end of the article © 2013 Vernet-der Garabedian et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Vernet-der Garabedian et al. Journal of Neuroinflammation 2013, 10:65 Page 2 of 11 http://www.jneuroinflammation.com/content/10/1/65 Background base-pair change [9]. Within the cerebellum, GluR∂2is Central nervous system (CNS) inflammation may de- present specifically in Purkinje cells [10,11]. The expres- velop in response to brain injury or in association with sion of the mutated allele leads to a cell-autonomous neurodegeneration in human neurodegenerative dis- process of Purkinje cell degeneration occurring between eases, such as Parkinson’s, Huntington’s and Alzheimer’s P7 and P30 [8]. As a result, 90% of the granule cells and diseases. Inflammation resulting from trauma or ische- 60 to 75% of the inferior olivary neurons die within the mia reperfusion injury includes endothelial damage, the first 3 months after birth, due to the loss of their target release of proinflammatory mediators, changes in vascu- [10,11]. The mechanisms by which Purkinje cells die lar permeability, infiltration with peripheral inflamma- have remained elusive. Activated caspase-3, DNA frag- tory cells and the activation of microglia, the innate mentation and autophagosomes in dying Purkinje cells immune cells of the brain, and astrocytes. It is marked were thought to be the results of the activation of by the recruitment of peripheral blood cells and the various cell-death execution pathways [12-14]. Recent formation of a scar. CD95 (Fas, APO-1), which was in vitro and in vivo studies supported a necrosis mech- initially identified as a death receptor, and its cognate anism caused by excitotoxicity and accompanied with ligand (CD95L, Fas-L) are both expressed in the brain. features of autophagy [15]. In the Grid2Lc/+ cerebellum, They are thought to contribute to the inflammatory this degenerative process takes place in an intact brain reaction through non-apoptotic signaling, mediating with a fully functional blood–brain barrier (N Delhaye- proinflammatory responses and triggering the recruit- Bouchaud and J Mariani, unpublished data). ment of peripheral macrophages and leukocytes to the The glial reaction to neuron loss has remained largely site of inflammation [1,2]. Recent data obtained by unexplored in the Grid2Lc/+ cerebellum. The presence Letellier et al. [3] for the spinal cord lesion model indi- of reactive astrocytes and high IL-1ß concentrations cate that the expression of CD95L on peripheral myeloid reported in previous studies suggest that an inflamma- cells plays a crucial role in the neural cell reaction at the tory process occurs in the Grid2Lc/+ cerebellum [16,17]. lesion. These authors demonstrated that peripheral cell In this study, we investigated the possible role of the CD95L expression, which is upregulated by the lesion, is CD95/CD95L system in the mechanism of neuronal required for the recruitment of peripheral myeloid cells death and in the inflammatory response, as an intrinsic to the injured tissue and contributes to neural cell loss component of the glial reaction in the Grid2Lc/+ cerebel- by acting on peripheral myeloid cells, rather than via lum. We analyzed the relationship between the death of direct CD95-induced apoptosis in CD95-bearing resi- cerebellar neurons and glial activation. Correlations dent neural cells [3]. In neurodegenerative diseases, the between glial cell activation and CD95 and CD95L ex- glial cell activation associated with neurodegeneration is pression on the one hand, and neurodegeneration events chronic. The inflammatory reaction is mild and features occurring from birth into adulthood on the other, indi- an absence of leukocytes within the neural parenchyma cate that the CD95/CD95L system is involved in the tissue; the possible occurrence of peripheral monocyte inflammatory reaction, mostly through astrocytes. infiltration remains a matter of debate [4-6]. Despite these important differences between the two types of Materials and methods inflammatory reaction, the CD95/CD95L system has Animals long been considered to play similar roles in the two Heterozygous Grid2Lc/+ mutant mice (Lc/+) and the corre- situations [1,7]. However, in the absence of infiltrating sponding wild-type strain, C57BL/6J X CBA (+/+), were peripheral cells, the contribution of the CD95/CD95L obtained by crossing Lc/+ males with +/+ females. Mutant system to neural cell reactions (neural degeneration offspring were identified on the basis of ataxic behavior at and the glial cell inflammatory reaction) remains about P15, or by genotyping for younger animals, as previ- unclear. Natural mutant mice, such as the ataxic ously described [12]. Animals were cared for and killed in Lurcher (Grid2Lc/+) mouse, which displays cerebellar accordance with the guidelines of the French National neurodegeneration, are valuable for investigations of Ethics Committee for Life and Health Sciences. the role of the CD95/CD95L system in this context making use of our extensive knowledge of cerebellar Glial cell cultures neurodegeneration in this mutant [8]. High-purity cultures of astrocytes and microglia were Ataxia becomes clinically detectable in the Lurcher prepared from mice from 5- and 1-day-old Lurcher mouse at about postnatal day 15 (P15). It results from litters, respectively, as previously described [18]. the progressive postnatal loss of cerebellar neurons due to a mutation in the ∂2 glutamate receptor subunit Tissue collection (GluR∂2) gene (Grid2). The Lurcher mutation is a Cerebella and cerebral cortices were collected on ice in semi-dominant gain-of-function mutation involving a an RPMI bath. The visible meninges and vessels were Vernet-der Garabedian et al. Journal of Neuroinflammation 2013, 10:65 Page 3 of 11 http://www.jneuroinflammation.com/content/10/1/65 stripped away under a binocular microscope [17]. The acidic protein (GFAP) labeling, sections prepared as dissected tissue was then immediately frozen in liquid described above were washed three times in TBS and nitrogen and stored at −80°C until use. incubated in TBS supplemented with 1% Triton X-100 and 10% normal goat serum for 20 minutes. Rabbit Immunoblotting polyclonal anti-GFAP antibody (Sigma;