Activation of the JNK-c-Jun pathway during the early phase of neuronal apoptosis induced by PrP106-126 and prion infection.

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.

Cerebellar Purkinje cell loss in aging Hu-Bcl-2 transgenic mice.

The number of cerebellar Purkinje cells is increased by over 40% in young transgenic mice that overexpress a human Bcl-2 transgene (Hu-Bcl-2). To determine whether the Bcl-2-mediated rescue of Purkinje cells persists through life, the numbers of Purkinje cells were estimated in 6-, 12-, 18-, and 24-month-old Hu-Bcl-2 transgenic mice and age-matched controls. In addition, the expression of four markers for Purkinje cell differentiation, calbindin (CaBP), the 67-kDa isoform of glutamic acid decarboxylase (GAD67), gamma-aminobutyric acid transaminase (GABA-T), and the NMDA-R1 receptor subtype (NMDA-NR1) was analyzed in 6-month-old Hu-Bcl-2 transgenics and controls to determine whether overexpression of Bcl-2 and rescue from naturally occurring cell death affects the normal differentiation of Purkinje cells. The estimates of Purkinje cell numbers showed that the number of Purkinje cells in the Hu-Bcl-2 transgenics declines after 6 months to approach wild-type values by 18 months. Although the exogenous human BCL-2 is still expressed in Purkinje cells at 24 months, the expression levels of human BCL-2 appear to decline significantly after 6 months, suggesting that survival of the supernumary Purkinje cells depends on the sustained overexpression of Bcl-2. All the Purkinje cells in the Hu-Bcl-2 transgenic mice appeared to express normal levels of the differentiation markers analyzed so there was no evidence for a class of Purkinje cells that do not differentiate normally when rescued from naturally occurring cell death.

Increased ischemia-induced angiogenesis in the staggerer mouse, a mutant of the nuclear receptor Roralpha.

Ror alpha is an orphan nuclear receptor. In homozygous staggerer mutant mice (Ror alpha(sg/sg)), a deletion within the Ror alpha gene leads to an overexpression of inflammatory cytokines. Because inflammation and hypoxia are 2 key stimuli of ischemia-induced angiogenesis, we studied the role of Ror alpha in this setting. Ischemia was induced by ligation of the right femoral artery in C57BL/6 Ror alpha(+/+) and Ror alpha(sg/sg) mice. After 3 and 28 days, angiogenesis was evaluated by microangiography, measurement of capillary density using immunohistochemistry (anti-CD31), and measurement of blood flow by laser Doppler imaging. At day 3, angiographic score and blood flow were similar in Ror alpha(sg/sg) mice and in Ror alpha(+/+) littermates. Conversely, at day 28, Ror alpha(sg/sg) mice showed a significant 2-fold increase in angiographic score and a 3-fold increase in capillary density within the ischemic hindlimb compared with control. Functionally, this coincided with a significant rise in leg perfusion in Ror alpha(sg/sg) mice (0.83+/-0.05 for ischemic/nonischemic leg perfusion ratio) compared with Ror(+/+) mice (0.66+/-0.04, P<0.05). In addition, more extensive angiogenesis in Ror alpha(sg/sg) mice correlated with an increased expression of eNOS protein by 83+/-12% and 71+/-24% at 3 and 28 days, respectively (P<0.05), whereas the level of the antiangiogenic cytokine IL-12 was significantly reduced by 38+/-10% at day 28 (P<0.05). Conversely, no changes in VEGF expression were observed. Our study identifies for the first time a new role for Ror alpha as a potent negative regulator of ischemia-induced angiogenesis.

Ceramide-induced apoptosis in cortical neurons is mediated by an increase in p38 phosphorylation and not by the decrease in ERK phosphorylation.

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.

Transgenic mice with neuronal overexpression of bcl-2 gene present navigation disabilities in a water task.

In the CNS, Bcl-2 is an antiapoptotic gene involved in the regulation of neuronal death. Transgenic mice overexpressing the human gene Bcl-2 (Hu-bcl-2 mice) showed delayed acquisition in two tasks requiring them to find a hidden platform starting from either a random or a constant starting location. The same mice were not deficient in another task requiring them to find a visible platform suggesting that the delay observed was not due to motor, visual or motivational deficits in the water. The delay observed in Hu-bcl-2 mice was more important in the random starting test in which the allocentric demand for navigation was stronger. The results suggested that allocentric navigation is particularly sensitive to abnormal CNS maturation following the overexpression of the bcl-2 gene. The specific deficits (motor learning, fear-related behavior and allocentric navigation) observed in Hu-bcl-2 mice suggest that the regulation of developmental neuronal death is crucial for multisensorial learning and emotional behavior.

Central origin of IL-1beta produced during peripheral inflammation: role of meninges.

Brain expression of Interleukin-1beta (IL-1beta) and its modulation have been extensively documented in different animal models. The majority of the studies were based on techniques such as RT-PCR, RIA and ELISA using global extracts. Meningeal tissue or choroid plexus both belong to the peripheral compartment. Thus, their presence in nervous tissue extracts may lead to erroneous interpretations. We measured IL-1beta mRNA in the cerebellum, hippocampus and cerebral cortex collected with meninges and in the same structures collected without meninges after a peripheral lipopolysaccharide (LPS) stimulation (0.5 microgram/g body weight), using RT-PCR. The presence of meninges in the extracts dramatically increased IL-1beta mRNA levels after LPS treatment while basal levels (before LPS injection) were not affected. Indeed, two-thirds of the response originated from the meningeal tissue and choroid plexus. Immunohistochemical studies showed that IL-1beta labelled cells, identified as macrophages, were exclusively localized in the ventricular and meningeal spaces, in our LPS condition treatment. These results point out the need of integrated analyses of data obtained with different techniques to demonstrate the presence in the nervous tissue of a molecule which is also widely expressed in the peripheral compartment.

Cerebellar Purkinje cell loss during life span of the heterozygous staggerer mouse (Rora(+)/Rora(sg)) is gender-related.

The staggerer mutation causes dysgenesis of the cerebellar cortex in the homozygous mutant (Rora(sg)/Rora(sg)). The mutation acts intrinsically within the Purkinje cells (PCs), leading to cytological abnormalities and a severe deficit in the number of these cells. In contrast, in the heterozygous staggerer (Rora(+)/Rora(sg)), the cytoarchitecture of the cerebellar cortex appears to be normal, but quantitative studies have revealed a significant loss of cerebellar neurons with advancing age. In the heterozygous reeler (+/rl), another mutant presenting a PC loss with age, we have found that only males were affected (Hadj-Sahraoui et al., 1996). In the present study, we have investigated whether a similar gender effect exists in the heterozygous staggerer during life span. PCs were counted on cerebellar sagittal sections in male and female Rora(+)/Rora(sg) and in their Rora(+)/Rora(+) littermates at 1, 3, 9, 13, 18, and 24 months of age. In the Rora(+)/Rora(+), the number of PCs remained stable until 18 months, but there was a 25% significant loss in 24- month-old mice of both genders. During life span, Rora(+)/Rora(+) males had slightly more PC than females. In the Rora(+)/Rora(sg) of both genders, the deficit in PC number was similar at 13 months but it appeared earlier in males, beginning between 1 and 3 months, and was aggravated regularly up to 13 months. By contrast, the decline was delayed and more abrupt in Rora(+)/Rora(sg) females, from a value still normal at 9 months to its maximal extent at 13 months. In view of these results, the heterozygous (Rora(+)/Rora(sg)) mouse offers an interesting model to test the interaction between sex, age, and genetic background on the development and maintenance of cerebellar neuronal populations.

Abnormal IL-1beta cytokine expression in the cerebellum of the ataxic mutant mice staggerer and lurcher.

To assess the extent to which interleukin-1beta (IL-1beta) may contribute to the development and/or progression of neurodegenerative processes, we have examined the levels of IL-1beta in the brain of two types of neurological mutant mice, staggerer and Lurcher. Using a quantitative immunological method (enzyme-linked immunosorbent assay, ELISA), we measured IL-1beta in the cerebellum, hippocampus and cerebral cortex of mutant mice at baseline and after peripheral LPS treatment. Two types of IL-1beta expression abnormalities were found in the mutant cerebella: higher basal level in Lurcher and a response to peripheral administration of LPS in staggerer. The association of IL-1beta expression abnormalities with the only brain structure where a massive neurodegeneration occurs supports the role of proinflammatory cytokines in this process.

Fear decrease in transgenic mice overexpressing bcl-2 in neurons.

Neuronal destruction in the amygdala, hypothalamus and cerebellum provokes a diminution in anxiety and neophobia. In transgenic mice that express the human bcl-2 gene under the control of neuron specific enolase promotor (Hu-bcl-2), BCL-2 overexpression reduces the naturally occurring neuronal death, producing an increase of the number of neurons and brain size. Since BCL-2 over-expression has been observed in different parts of the brain and especially in the amygdaloid nuclei, the hypothalamus and the cerebellum, we studied the fear-related behavior of these transgenic mice. Hu-bcl-2 transgenic mice showed a decrease in anxiety and neophobia, indicating that, for this particular behavior, supernumerary neurons elicit the same modification as that observed after neuronal destruction.

Over-expression of interleukin-1 beta-converting enzyme mRNA in staggerer cerebellum.

Interleukin-1 beta-converting enzyme (ICE), involved in the maturation process of interleukin-1 beta (IL-1 beta, is a homologue of ced-3, a protease required for programmed cell death in Caenorhabditis elegans. Over-expression of ICE induces programmed cell death in certain mammalian cell types, whereas in neurones of the central nervous system such a role has yet to be established. We show that ICE mRNA expression is increased 4-fold in the cerebellum of homozygous staggerer mice, where IL-1 beta mRNA is overexpressed and programmed neuronal cell death occurs. Intraperitoneal injection of endotoxin (LPS) induced a strong phasic increase in IL mRNA levels in the cerebellum, whereas the ICE mRNA level increased only moderately. Involvement of ICE in neuronal cell death in the cerebellum of staggerer mice is suspected.

Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis.

In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogenous uridine and 4-thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine. On a high-Mg2+ sucrose gradient, 33 +/- 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4-5% substitution level (40 thiouridine molecules/particle). Surprisingly, 3-4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA. Specific 366-nm photoactivation increased this proportion to 10-12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross-linking proceeds with apparent first-order kinetics with a quantum yield close to 5 X 10(-3). Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.

Structure of a murine alpha interferon pseudogene with a repetitive R-type sequence in the 3' flanking region.

A murine alpha interferon pseudogene was identified in a mouse genomic library. The nucleotide sequence revealed several in-phase termination codons within the gene and repetitive oligonucleotides in the flanking regions. The nucleotide sequences and the amino acids of the peptide signal sequences were compared with known human alpha interferon genes and the pseudogene.