Jean Mariani

The orphan nuclear receptor RORα is a negative regulator of the inflammatory response

Retinoid‐related orphan receptor α (RORα) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the RORα gene, suffer from immune abnormalities, we generated an adenovirus encoding RORα1 to investigate its potential role in control of the inflammatory response. We demonstrated that RORα is expressed in human primary smooth‐muscle cells and that ectopic expression of RORα1 inhibits TNFα‐induced IL‐6, IL‐8 and COX‐2 expression in these cells. RORα1 negatively interferes with the NF‐κB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of RORα1 on NF‐κB is associated with the induction of IκBα, the major inhibitory protein of the NF‐κB signalling pathway, whose expression was found to be transcriptionally upregulated by RORα1 via a ROR response element in the IκBα promoter. Taken together, these data identify RORα1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis.

Severe Atherosclerosis and Hypoalphalipoproteinemia in the Staggerer Mouse, a Mutant of the Nuclear Receptor RORα

BACKGROUND Hypoalphalipoproteinemia is the most common lipoprotein abnormality in patients with coronary artery disease, yet its causes are unknown. METHODS AND RESULTS We show that the homozygous staggerer (sg/sg) mutant mouse, which carries a deletion within the nuclear receptor RORalpha gene, develops severe atherosclerosis when maintained on an atherogenic diet. In addition, sg/sg mice display a profound hypoalphalipoproteinemia, which is associated with decreased plasma levels of the major HDL proteins, apolipoprotein (apo) A-I and apoA-II. This decrease in HDL levels in sg/sg mice is due to lowered apoA-I gene expression in the intestine but not in the liver. ApoA-II gene expression is unaffected. CONCLUSIONS These results suggest that the RORalpha gene contributes to the plasma HDL level and susceptibility to atherosclerosis.

Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.

Hypoxia–ischaemia in the developing brain results in brain injury with prominent features of apoptosis. In the present study, a third generation dipeptidyl broad‐spectrum caspase inhibitor, quinoline‐Val‐Asp(Ome)‐CH2‐O‐phenoxy (Q‐VD‐OPh), was tested in a model of unilateral focal ischaemia with reperfusion in 7‐day‐old rats. Q‐VD‐OPh (1 mg/kg, i.p.) reduced cell death, resulting in significant neuroprotection at 48 h of recovery (infarct volume of 12.6 ± 2.8 vs. 24.3 ± 2.2%, p = 0.006). The neuroprotective effects observed at 48 h post‐ischaemia hold up at 21 days of survival time and attenuate neurological dysfunction. Analysis by gender revealed that females were strongly protected (6.7 ± 3.3%, p = 0.006), in contrast to males in which there was no significant effect, when Q‐VD‐OPh was given after clip removal on the left common carotid artery. Immunoblot analysis demonstrated that Q‐VD‐OPh inhibits caspase 3 cleavage into its p17 active form and caspase 1 up‐regulation and cleavage in vivo. Following ischaemia in P7 rats, males and females displayed different time course and pattern of cytochrome c release and active p17 caspase 3 during the first 24 h of recovery. In contrast, no significant difference was observed for caspase 1 expression between genders. These results indicate that ischaemia activates caspases shortly after reperfusion and that the sex of the animal may strongly influences apoptotic pathways in the pathogenesis of neonatal brain injury. The specificity, effectiveness, and reduced toxicity of Q‐VD‐OPh may determine the potential use of peptide‐derived irreversible caspase inhibitors as promising therapeutics.

Synapse Elimination in the Central Nervous System: Functional Significance and Cellular Mechanisms

Recent research into the developmental elimination of supernumerary synapses has increased understanding of this process. In this review we discuss synapse elimination both at the neuromuscular junction and in the central nervous system, considering some possible underlying mechanisms suggested by recent studies. In addition a well-described example of central nervous system synapse elimination, the climbing fiber-Purkinje cell synapse of the cerebellum, is used to explore the functional significance of synaptic regression during brain development.

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.

Noncoding RNAs in Long-Term Memory Formation

Current research exploring the molecular basis of memory focuses mainly on proteins despite recent genomic studies reporting the abundant transcription of non-protein-coding RNA (ncRNA). Although ncRNAs are involved in a diverse range of biological processes, they are particularly prevalent within the nervous system, where they contribute towards the complexity and function of the mammalian brain. In this review, we apply recent advances in ncRNA biology to predict a critical role for ncRNAs in the molecular mechanisms underlying memory formation and maintenance. We describe the role of ncRNAs in regulating the translation, stability, and editing of mRNA populations in response to synaptic activity during memory formation and the role of ncRNAs in the epigenetic and transcriptional programs that underlie long-term memory storage. We also consider ncRNAs acting as an additional avenue of communication between neurons by their intercellular trafficking. Taken together, the emerging evidence suggests a central role for ncRNAs in memory formation and provokes novel research directions in this field. NEUROSCIENTIST 14(5):434—445, 2008. DOI: 10.1177/1073858408319187

Neurobiological effects of a null mutation depend on genetic context: comparison between two hotfoot alleles of the delta-2 ionotropic glutamate receptor

Hotfoot is a mutant mouse with an ataxic phenotype which has been shown to be due to a mutation in the Grid2 gene. In this paper, we compare molecular, morphological, electrophysiological and behavioral features of two Grid2 alleles: Grid2(ho-4J) and Grid2(ho-Nancy). We first show that these two mutations are deletions in the open reading frame of the gene and that no GRID2 protein is detectable in extracts of mutant cerebella, suggesting that the two alleles are null-like mutations. Morphological and electrophysiological analyses reveal no obvious differences between the two strains: both strains showed the naked Purkinje dendritic spines and mismatch between the length of the presynaptic active zone and postsynaptic differentiation characteristic of the hotfoot mutation; and the same low level (20%) of multiple climbing fiber innervation of Purkinje cells was found in both strains. Only differences in motor behavior were found between the two strains. The Grid2(ho-4J) mouse shows more severe ataxia that the Grid2(ho-Nancy) mouse and, although both strains show a clear capacity to improve their performance of a motor task with training, the Grid2(ho-4J) performance remains very poor whereas Grid2(ho-Nancy) mice approach control levels. The only difference between the two strains is their genetic background. Our results show that the genetic background must be taken into account when analyzing sensorimotor performances of mutant mice.

Increased cerebellar Purkinje cell numbers in mice overexpressing a human Bcl-2 transgene

The Purkinje cell is a primary organizer in the development of the cerebellum. Purkinje cells may provide positional information cues that regulate afferent innervation, and Purkinje cell target size controls the adult number of afferent olivary neurons and granule cells. While Purkinje cells are necessary for the survival of olivary neurons and granule cells during periods of programmed cell death, little is known about the survival requirements of Purkinje cells in vivo. To determine if Purkinje cells are subject to programmed cell death during development we have analyzed Purkinje cell numbers in two lines of transgenic mice that overexpress a human gene for bcl‐2 (Hu‐bcl‐2). Bcl‐2 is a protooncogene that inhibits apoptosis in many cell types. Overexpression of bcl‐2 in vitro and in vivo rescues neurons from trophic factor deprivation or naturally occurring cell death. In the mice analyzed in this study, transgene expression is driven by the neuron‐specific enolase promoter that is first expressed embryonically in most regions of the brain in one line and postnatally in the second line. We have counted Purkinje cells in three adult control mice, five early overexpressing transgenics, and three late expressing transgenics. The number of Purkinje cells in the Hu‐bcl‐2 transgenic mice is significantly increased above control numbers, with an increase of 43% in the embryonically overexpressing line and an increase of 27% in the postnatally overexpressing line. Because bcl‐2 overexpression has been shown to rescue other neurons from programmed cell death, the increase in Purkinje cell numbers in overexpressing bcl‐2 transgenics suggests that Purkinje cells undergo a period of cell death during normal development.

NMDA RECEPTOR CONTRIBUTION TO THE CLIMBING FIBER RESPONSE IN THE ADULT MOUSE PURKINJE CELL

Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at ∼21 d after birth and control the complex spike waveform.

Impaired Sequential Egocentric and Allocentric Memories in Forebrain-Specific–NMDA Receptor Knock-Out Mice during a New Task Dissociating Strategies of Navigation

The hippocampus is considered to play a role in allocentric but not in egocentric spatial learning. How does this view fit with the emerging evidence that the hippocampus and possibly related cortical areas are necessary for episodic-like memory, i.e., in all situations in which events need to be spatially or sequentially organized? Are NMDA receptor-dependent mechanisms crucial for the acquisition of spatiotemporal relationships? To address this issue, we used knock-out (KO) mice lacking hippocampal CA1 NMDA receptors and presenting a reduction of these receptors in the deep cortical layers (NR1-KO mice). A new task (the starmaze) was designed, allowing us to distinguish allocentric and sequential-egocentric memories. NR1-KO mice were impaired in acquiring both types of memory. Our findings suggest that memories composed of multiple spatiotemporal events require intact NMDA receptors-dependent mechanisms in CA1 and possibly in the deep cortical layers.