Ceramide, the central molecule of the sphingomyelin pathway, serves as a second messenger for cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis. In this study we show that c2-ceramide induces apoptosis in primary cortical neuron cultures and that this effect correlates with differential modulation of mitogen-activated protein kinase (MAPK) cascades. Phosphorylation of extracellular signal-regulated kinases (ERKs) and their upstream activators MAPK kinases (MEKs), as measured by immunoblotting is rapidly decreased by c2-ceramide. However, the MEK inhibitor PD98059 alone does not induce apoptosis and in combination with c2-ceramide it does not modify c2-ceramide-induced apoptosis. Treatment with c2-ceramide increases p38 and c-Jun N-terminal kinase (JNK) phosphorylation before and during caspase-3 activation. The p38 inhibitor SB203580 partially protects cortical neurons against c2-ceramide-induced apoptosis, implicating the p38 pathway in this process. The c2-ceramide treatment also increases levels of c-jun, c-fos and p53 mRNA in primary cortical neuron cultures, but this is independent of p38 activation. Our study further elucidates the time-courses of MAPK cascade modulation, and of c-jun, c-fos and p53 activation during c2-ceramide-induced neuronal apoptosis. It reveals that one of the activated kinases, p38, is necessary for this apoptosis.

Retinoic acid receptor-related orphan receptor a (RORa) is a transcription factor belonging to the superfamily of nuclear receptors. Disruption of the Rora gene in the mouse results in a defect in the development of Purkinje cells leading to a cerebellar atrophy, which suggests a neuroprotective role for RORa. To test this hypothesis, the survival rate of lentiviralmediated human RORa1-overexpressing neurones has been evaluated in response to different stressors disturbing the redox homeostasis, such as b-amyloid peptide, c 2 -ceramide and H 2 O 2 . We show that overexpression of human RORa1 provides neuroprotection by increasing the expression of the antioxidant proteins glutathione peroxidase 1 and peroxiredoxin 6, leading to a reduction in the accumulation of stressinduced reactive oxygen species. We further demonstrate that the neuroprotective effect of RORa is predominantly mediated by glutathione peroxidase 1 and peroxiredoxin 6. These results suggest a new role for RORa in the control of the neuronal oxidative stress and thus represents a new transcription factor of interest in the regulation of reactive oxygen species-induced neurodegenerative processes during ageing. Keywords: apoptosis, lentivirus, neuroprotection, oxidative stress, retinoic acid receptor-related orphan receptor a. J. Neurochem. 96, 1778-1789.There is accumulating evidence that a proper balance between oxidants and antioxidants is of systemic importance for health and longevity. Homeostasis of redox status is normally maintained by intracellular regulatory molecules and enzymes such as glutathione, thioredoxin, catalase, superoxide dismutase and peroxidases. Exposure to oxidants can lead to oxidative stress wherein the antioxidant status of the cell is thrown out of balance and may lead to a variety of symptoms including apoptosis. How the expression of these enzymes, implicated in homeostasis of redox status, is regulated and which factors are involved is still poorly understood.Retinoic acid receptor-related orphan receptor a (RORa) is a member of the superfamily of the nuclear receptors (Giguere 1999). Initially described as an orphan receptor, RORa had long been considered a constitutive activator of transcription in the absence of exogenous ligand (Atkins et al. 1999;Harris et al. 2002). Recently, however, cholesterol and 7-dehydrocholesterol were shown to be natural ligands of the transcription factor RORa (Kallen et al. 2002;Bitsch et al. 2003).Mutant mice lacking functional RORa protein (Hamilton et al. 1996;Steinmayr et al. 1998) develop a severe cerebellar ataxia due to a massive cerebellar neurodegeneration (Sidman et al. 1962 Abbreviations used: Ab, b-amyloid peptide; GFP, green fluorescent protein; Gpx, glutathione peroxidase; hRORa, human retinoic acid receptor-related orphan receptor a; Prx, peroxiredoxin; RORa, retinoic acid receptor-related orphan receptor a; ROS, reactive oxygen species; si-RNA, small interfering RNA.

Prion diseases are neurodegenerative pathologies characterized by apoptotic neuronal death. Although the late execution phase of neuronal apoptosis is beginning to be characterized, the sequence of events occurring during the early decision phase is not yet well known. In murine cortical neurons in primary culture, apoptosis was first induced by exposure to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP106-126. Exposure to its aggregated form induced a massive neuronal death within 24 h. Apoptosis was characterized by nuclear fragmentation, neuritic retraction and fragmentation and activation of caspase-3. During the early decision phase, reactive oxygen species were detected after 3 h. Using immunocytochemistry, we showed a peak of phosphorylated c-Jun-N-terminal kinase (JNK) translocation into the nucleus after 8 h, along with the activation of the nuclear c-Jun transcription factor. Both pharmacological inhibition of JNK by SP600125 and overexpression of a dominant negative form of c-Jun significantly reduced neuronal death, while the MAPK p38 inhibitor SB203580 had no effect. Apoptosis was also studied after exposure of tg338 cortical neurons in primary culture to sheep scrapie agent. In this model, prion-induced neuronal apoptosis gradually increased with time and induced a 40% cell death after 2 weeks exposure. Immunocytochemical analysis showed early c-Jun activation after 7 days. In summary, the JNK-c-Jun pathway plays an important role in neuronal apoptosis induced by PrP106-126. This pathway is also activated during scrapie infection and may be involved in prion-induced neuronal death. Pharmacological blockade of early pathways opens new therapeutic prospects for scrapie PrP-based pathologies.