Laurent Bezin

ISG20L2, a Novel Vertebrate Nucleolar Exoribonuclease Involved in Ribosome Biogenesis

Proteomics analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present study describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3′ to 5′ exoribonuclease involved in ribosome biogenesis at the level of 5.8 S rRNA maturation, more specifically in the processing of the 12 S precursor rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Identification of the binding partners of ISG20L2 confirmed its involvement in the biogenesis of the large ribosomal subunit. These results strongly support the notion that, in human, as it was demonstrated in yeast, 5.8 S rRNA maturation requires several proteins in addition to the exosome complex. Furthermore this observation greatly sustains the idea that the extremely conserved need for correctly processed rRNAs in vertebrates and yeast is achieved by close but different mechanisms.

Bax, Bcl-2, and cyclin expression and apoptosis in rat substantia nigra during development

Naturally occurring cell death via apoptosis has been reported in the substantia nigra of rats during development, culminating during the perinatal period. Cellular pathways leading to apoptotic death of developing nigral dopamine neurons remain unknown, although the apoptotic mediator, caspase 3, has been shown to be activated during this process. Our previous results demonstrated the inability of antioxidants to rescue the nigral dopamine neurons that undergo apoptosis during development. In the present study, we investigated using immunohistochemistry the expression of cyclins D1, D3, and E in the substantia nigra during pre- and postnatal development, since their re-expression in postmitotic neurons has been proposed to contribute to developmental apoptosis. We also investigated by Western blot analysis of nigral tissue isolated during the first postnatal week the expression of the anti- and pro-apoptotic proteins, Bcl-2 and Bax, respectively, since altered Bcl-2 expression during developmental apoptosis has been described. During apoptotic death of nigral dopamine neurons in development, we detected a significant increase in the Bax:Bcl-2 ratio, which is consistent with enhanced apoptosis. There were no changes in the expression of the cyclins during the same apoptotic period. These novel findings suggest that nigral dopamine neurons undergo developmental apoptotic death through a Bax:Bcl-2-sensitive pathway that does not involve cyclin mediation.

Singular subsets of locus coeruleus neurons may recover tyrosine hydroxylase phenotype transiently expressed during development

The number of tyrosine hydroxylase (TH)-expressing neurons appears to be precisely determined in basal conditions within the noradrenergic pontine nucleus locus coeruleus (LC). However, additional neurons exhibiting TH phenotype have been observed in the adult rat LC following a single administration of RU 24722, a potent inducer of TH expression specific to the LC. The neurons acquiring TH phenotype following treatment had a topographical localization similar to that of the neurons, which transiently expressed TH during postnatal development and lost TH phenotype during the third postnatal week. The idea that the fluctuation of TH phenotype in singular subsets of LC neurons during development may be selectively restored in adults is of particular interest. The present study attempted to determine whether the cells in which TH expression was repressed during the third postnatal week could correspond to those which exhibited TH phenotype in response to RU 24722 treatment in adults. We first verified that no massive cell death occurred in the LC during the period ranging from days 13 to 30. Then, we observed that both cell populations exhibited the same altered steady-state concentration of TH-mRNA as compared to cells that permanently expressed TH. Finally, we demonstrated the presence of TH-negative neurons expressing the homeodomain transcription factor Phox2a, specific for the determination of noradrenergic phenotype, providing further evidence that resting-noradrenergic neurons exist in the adult rat LC under basal conditions. These neurons provide interesting prospective for gain of noradrenergic function when classical noradrenergic LC neurons are impaired.

Unexpected expression of orexin-B in basal conditions and increased levels in the adult rat hippocampus during pilocarpine-induced epileptogenesis

Orexin-A (OX-A) and -B (OX-B) peptides present in the hippocampus are considered to be exclusively contained in fibers arising from hypothalamus neurons, which were established as the only source of orexins (OXs). Because OX-A is known to exert excitatory actions in the hippocampus, we hypothesized that the level of OXs targeted toward the hippocampus may be increased following status-epilepticus (SE)-induced epileptogenesis in the rat pilocarpine model of temporal lobe epilepsy. We found that tissue concentration of prepro-OX mRNA, which encodes for both peptides, rapidly decreased in the hypothalamus of rats having experienced pilocarpine-induced SE (Pilo-SE) followed by a reduced density of OX-A and OX-B immunopositive fibers arising from these neurons. By contrast, it was unexpected to detect within the hippocampus the presence of prepro-OX mRNA in basal conditions and to evidence its up-regulation during the 1- to 3-day period following Pilo-SE. The number of prepro-OX mRNA copies determined by real-time RT-PCR was approximately 50-fold lower in the hippocampus than that in the hypothalamus, precluding the use of in situ hybridization to localize the cells which synthesize the transcript within the hippocampus. The increase in prepro-OX mRNA level within the hippocampus was accompanied by the detection of OX-B-like immunoreactivity 2-3 days post-SE, not only in pyramidal neurons, granule cells and cell bodies resembling interneurons, but also in some astrocytes scattered throughout the hippocampus. The present data suggest that the gene encoding OXs can be activated in the hippocampus, which may play a role in the pathogenesis of epilepsy.

Stochastic Loss of Silencing of the Imprinted Ndn/NDN Allele, in a Mouse Model and Humans with Prader-Willi Syndrome, Has Functional Consequences

Genomic imprinting is a process that causes genes to be expressed from one allele only according to parental origin, the other allele being silent. Diseases can arise when the normally active alleles are not expressed. In this context, low level of expression of the normally silent alleles has been considered as genetic noise although such expression has never been further studied. Prader-Willi Syndrome (PWS) is a neurodevelopmental disease involving imprinted genes, including NDN, which are only expressed from the paternally inherited allele, with the maternally inherited allele silent. We present the first in-depth study of the low expression of a normally silent imprinted allele, in pathological context. Using a variety of qualitative and quantitative approaches and comparing wild-type, heterozygous and homozygous mice deleted for Ndn, we show that, in absence of the paternal Ndn allele, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. The level of this expression is sex-dependent and shows transgenerational epigenetic inheritance. In about 50% of mutant mice, this expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. In wild-type brains, the maternal Ndn allele is never expressed. However, using several mouse models, we reveal a competition between non-imprinted Ndn promoters which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn allelic exclusion occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Our data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS. Overall our results reveal high non-genetic heterogeneity between genetically identical individuals that might underlie the variability of the phenotype.

Controlled targeting of tyrosine hydroxylase protein toward processes of locus coeruleus neurons during postnatal development.

Dendrites of locus coeruleus (LC) neurons laying within the pericoerulean neuropil (PCA) organize the major site where tyrosine hydroxylase (TH) is present throughout postnatal development. Those dendrites constitute the neuronal compartment in which TH levels increase beyond postnatal day (P) 21 or after RU24722-induced TH expression. Distal LC dendrites are present in the PCA by at least P20 but are devoid of TH and can rapidly accumulate TH protein when gene induction is triggered. Contrasting with the increase in TH levels within LC perikarya and dendrites, TH-mRNA concentration remains constant in LC perikarya from P4 to P42. Thus, supposing TH synthesis and degradation are also constant, any change in TH levels targeted toward axons might be balanced by a shift in the TH deposition within LC dendrites. This mechanism may be crucial in functions that the different processes of LC neurons have at critical steps of postnatal ontogeny.

Enhanced tail pinch-induced activation of catecholamine metabolism in the pericerulean area of RU 24722-treated rats.

Our study was devoted to determine in freely moving rats whether the increase in tissue concentration of tyrosine hydroxylase (TH) elicited by a single administration of RU 24722 could modify the catecholaminergic reactivity of neuronal processes present in the rostrolateral part of the pericerulean area (r-lPCA) in response to tail pinch. Catecholaminergic activity was monitored by measuring in vivo the concentration of dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) using microdialysis coupled to HPLC detection. In this study, the microdialysis probe was implanted at a sufficient distance from the lateral border of rostral nucleus locus ceruleus (LC) to avoid a large contribution of the noradrenergic cell bodies in the measurements performed. We first evidenced that DOPAC measured in the r-lPCA indicated the functional state of catecholaminergic metabolism in neuronal processes (dendrites and fibers) laying in this region. We also showed that the enhancement of TH protein concentration in the r-lPCA following RU 24722 treatment supported an increased in vivo catecholaminergic metabolism in this region. Furthermore, catecholaminergic metabolism response to tail pinch was potentiated in animals with greater TH tissue concentration. Thus, our study reveals that the modulation of both TH concentration and catecholaminergic metabolism in the r-lPCA may be critical in the functioning of cells and neuronal elements present in this region, notably in adaptive responses to noxious stimuli.

Inhibition of Oxidative Stress during Developmental Cell Death: Cellular and Behavioral Effects

Dopamine neurons in the substantia nigra during development undergo cell death, which has been described as apoptotic (1,2), with the main apoptotic peak occurring during the perinatal period (1). Several studies have investigated the cellular mechanisms underlying the apoptosis of these neurons, but the precise mechanisms are still poorly understood. In cultured catecholamine cells, tetrahydrobiopterin (BH4) has been shown to participate in the apoptotic cell death process (3). Both dopamine and BH4 metabolism can produce reactive oxygen species, which can cause cellular damage including lipid peroxidation, which is characterized by the formation of lipid byproducts derived from the breakdown of polyunsaturated fatty acids and related esters (6). Reactive oxygen species are actively scavenged by antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). (−)-Deprenyl is an irreversible inhibitor of monoamine oxidase-B, which has been shown to prevent apoptosis of cultured fetal dopamine neurons through its antioxidant properties (7,8). α-Tocopherol (vitamin E) is a lipophilic antioxidant that can prevent lipid peroxidation, and has been used to reduce neuronal damage induced by neurotoxic agents (9). We have previously shown that deprenyl and α-tocopherol had no protective effects against developmental apoptosis of dopamine neurons (4,5). In present report, we investigated the effects of deprenyl and α-tocopherol on levels of antioxidant enzymes, lipid oxidation, and the locomotor and learning behaviors of rats.