PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

Transforming growth factor alpha promotes sequential conversion of mature astrocytes into neural progenitors and stem cells.

An instability of the mature cell phenotype is thought to participate to the formation of gliomas, primary brain tumors deriving from astrocytes and/or neural stem cells. Transforming growth factor alpha (TGFalpha) is an erbB1 ligand overexpressed in the earliest stages of gliomas, and exerts trophic effects on gliomal cells and astrocytes. Here, we questioned whether prolonged TGFalpha exposure affects the stability of the normal mature astrocyte phenotype. We first developed astrocyte cultures devoid of residual neural stem cells or progenitors. We demonstrate that days of TGFalpha treatment result in the functional conversion of a population of mature astrocytes into radial glial cells, a population of neural progenitors. TGFalpha-generated radial glial cells support embryonic neurons migration, and give birth to cells of the neuronal lineage, expressing neuronal markers and the electrophysiological properties of neuroblasts. Lengthening TGFalpha treatment to months results in the delayed appearance of cells with neural stem cells properties: they form floating cellular spheres that are self-renewing, can be clonally derived from a single cell and differentiated into cells of the neuronal lineage. This study uncovers a novel population of mature astrocytes capable, in response to a single epigenetic factor, to regress progressively into a neural stem-like cell stage via an intermediate progenitor stage.

DOCK4 promotes loss of proliferation in glioblastoma progenitor cells through nuclear beta-catenin accumulation and subsequent miR-302-367 cluster expression.

Glioblastomas (GBM) are lethal primitive brain tumours characterized by a strong intra-tumour heterogeneity. We observed in GBM tissues the coexistence of functionally divergent micro-territories either enriched in more differentiated and non-mitotic cells or in mitotic undifferentiated OLIG2 positive cells while sharing similar genomic abnormalities. Understanding the formation of such functionally divergent micro-territories in glioblastomas (GBM) is essential to comprehend GBM biogenesis, plasticity and to develop therapies. Here we report an unexpected anti-proliferative role of beta-catenin in non-mitotic differentiated GBM cells. By cell type specific stimulation of miR-302, which directly represses cyclin D1 and stemness features, beta-catenin is capable to change its known proliferative function. Nuclear beta-catenin accumulation in non-mitotic cells is due to a feed forward mechanism between DOCK4 and beta-catenin, allowed by increased GSK3-beta activity. DOCK4 over expression suppresses selfrenewal and tumorigenicity of GBM stem-like cells. Accordingly in the frame of GBM median of survival, increased level of DOCK4 predicts improved patient survival.

Transforming growth factor alpha acts as a gliatrophin for mouse and human astrocytes.

Astrocyte death has been implicated in several neuropathological diseases, but the identification of molecules susceptible of promoting astrocyte survival has been elusive. We investigated whether transforming growth factor alpha (TGFalpha), an erbB1/EGFR ligand, which promotes glioma progression and affects astrocyte metabolism at embryonic and adult stages, regulates astrocyte survival. Primary serum-free astrocyte cultures from post-natal mouse and fetal human cortices were used. Transforming growth factor alpha protected both species of astrocytes from staurosporine-induced apoptosis. In serum-free medium, mouse astrocytes did not survive beyond 2 months while TGFalpha-treated astrocytes survived up to 12 months. Transforming growth factor alpha also promoted long-term survival of human astrocytes. We additionally extended TGFalpha proliferative effects to human astrocytes. After 3 days of permanent application, TGFalpha induced a major downregulation of both erbB1 and erbB2. This downregulation did not impair the functional activation of the receptors, as ascertained by their tyrosine phosphorylation and the continuous stimulation of both ERK/MAPK and PI3K/Akt pathways up to 7 days, the longest time examined. The full cellular effects of TGFalpha required activation of both transduction pathways. Enhanced proliferation and survival thus define TGFalpha as a gliatrophin for mammalian astrocytes. These results demonstrate that in normal, non-transformed astrocytes, sustained and functional erbBs activation is achieved without bypassing ligand-induced receptors downregulation.

Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptor-activated kinase by an adhesion-independent mechanism.

PEA-15 is a small, death effector-domain (DED)-containing protein that was recently demonstrated to inhibit tumor necrosis factor-alpha-induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5'-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.

Knock-out of the neural death effector domain protein PEA-15 demonstrates that its expression protects astrocytes from TNFalpha-induced apoptosis.

Apoptosis is a very general phenomenon, but only a few reports concern astrocytes. Indeed, astrocytes express receptors for tumor necrosis factor (TNF) alpha, a cytokine demonstrated on many cells and tissues to mediate apoptosis after recruitment of adaptor proteins containing a death effector domain (DED). PEA-15 is a DED-containing protein prominently expressed in the CNS and particularly abundant in astrocytes. This led us to investigate if PEA-15 expression could be involved in astrocytic protection against deleterious effects of TNF. In vitro assays evidence that PEA-15 may bind to DED-containing protein FADD and caspase-8 known to be apical adaptors of the TNF apoptotic signaling. After generation of PEA-15 null mutant mice, our results demonstrate that PEA-15 expression increases astrocyte survival after exposure to TNF.

Stathmin phosphorylation patterns discriminate between distinct transduction pathways of human T lymphocyte activation through CD2 triggering.

CD2 triggering of human T lymphocyte activation has been associated with the activation of different interacting protein kinases, including protein kinase C (PKC). However the precise roles of its phosphorylated substrates are still unknown. We show here that PKC-dependent and -independent pathways are responsible for the CD2-induced phosphorylation of stathmin, a ubiquitous soluble phosphoprotein, most likely acting as a general intracellular relay integrating various second messenger pathways. The phosphorylated variants of stathmin provide a fingerprint reflecting the second messenger pathway(s) stimulated. The respective roles of both PKC and stathmin in the regulation of T lymphocyte proliferation are discussed.

Genetic heterogeneity of autosomal dominant cerebellar ataxia type 1: clinical and genetic analysis of 10 French families.

We performed linkage analysis between the gene responsible for spinal cerebellar ataxia 1 (SCA1) and the highly polymorphic chromosome 6 locus, D6S89, in 10 French families with autosomal dominant cerebellar ataxia (ADCA) type 1. These families were clinically indistinguishable except for one family with loss of hearing and vision. Very close linkage was observed in four families, with no evidence of recombination between SCA1 and D6S89. Linkage with D6S89 was excluded in the six others, thus demonstrating genetic heterogeneity for ADCA type 1. The D6S89 marker, which is very closely linked to the disease locus, can be used to identify SCA1 families and will lead to predictive testing.

Cyclic AMP reduces adhesion of isolated neuronal growth cones from developing rat forebrain to an astrocytic cell line from embryonic mouse striatum.

We have recently shown that isolated neuronal growth cones from developing rat forebrain possess an appreciable activity of adenylate cyclase, producing cyclic adenosine monophosphate, which can be stimulated by various neurotransmitter receptor agonists and by forskolin [Lockerbie R. O., Hervé D., Blanc G., Tassin J. P. and Glowinski J. (1988) Devl Brain Res. 38, 19-25]. In the present study, we have investigated the effect of cyclic adenosine monophosphate in an in vitro adhesion assay established between [3H]GABA-labelled isolated growth cones and a Simian virus-40 transformed astrocytic cell line from embryonic mouse striatum. Adhesion of the isolated growth cones onto the astrocytic clone increased steadily up to about 45 min before it began to level off at ca 16-18% of total [3H]GABA-labelled isolated growth cones added. Adhesion of the isolated growth cones onto the astrocytic clone was much superior to that seen on polyornithine and, in particular, on non-treated tissue culture wells. Adhesion "at plateau" was independent of both temperature and extracellular Ca2+ and was markedly reduced (ca 50%) by trypsin pre-treatment of the isolated growth cones. Pre-treatment of the isolated growth cones with either forskolin or lipophilic analogues of cyclic adenosine monophosphate attenuated adhesion in a time- and concentration-dependent manner. Approximately 30% reduction in adhesion to the astrocytic clone "at plateau" was observed after a 15 min pre-treatment of the isolated growth cones with forskolin at 10(-4) M or cyclic adenosine monophosphate analogues at 10(-3) M. A cyclic guanosine monophosphate analogue was without effect on adhesion of isolated growth cones. Scanning electron microscope analysis showed that isolated growth cones pre-treated with either cyclic adenosine monophosphate analogues or forskolin had a simpler morphology when attached to the astrocytic clone than isolated growth cones under control conditions. Pre-treatment of the isolated growth cones with low concentrations of cyclic adenosine monophosphate analogues increased protein kinase activity, measured using an exogenous histone phosphate acceptor, to a level which could not be further stimulated by cyclic adenosine monophosphate. Pre-treatment with a cyclic guanosine monophosphate analogue produced the same effect but only at much higher concentrations than those required for cyclic adenosine monophosphate analogues.

The expression of PEA-15 (phosphoprotein enriched in astrocytes of 15 kDa) defines subpopulations of astrocytes and neurons throughout the adult mouse brain.

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is an abundant phosphoprotein in primary cultures of mouse brain astrocytes. Its capability to interact with members of the apoptotic and mitogen activated protein (MAP) kinase cascades endows PEA-15 with anti-apoptotic and anti-proliferative properties. We analyzed the in vivo cellular sources of PEA-15 in the normal adult mouse brain using a novel polyclonal antibody. Immunohistochemical assays revealed numerous PEA-15-immunoreactive cells throughout the brain of wild-type adult mice while no immunoreactive signal was observed in the brain of PEA-15 -/- mice. Cell morphology and double immunofluorescent staining showed that both astrocytes and neurons could be cellular sources of PEA-15. Closer examination revealed that in a given area only part of the astrocytes expressed the protein. The hippocampus was the most striking example of this heterogeneity, a spatial segregation restricting PEA-15 positive astrocytes to the CA1 and CA3 regions. A PEA-15 immunoreactive signal was also observed in a few cells within the subventricular zone and the rostral migratory stream. In vivo analysis of an eventual PEA-15 regulation in astrocytes was performed using a model of astrogliosis occurring along motor neurons degeneration, the transgenic mouse expressing the mutant G93A human superoxyde-dismutase-1, a model of amyotrophic lateral sclerosis. We observed a marked up-regulation of PEA-15 in reactive astrocytes that had developed throughout the ventral horn of the lumbar spinal cord of the transgenic mice. The heterogeneous cellular expression of the protein and its increased expression in pathological situations, combined with the known properties of PEA-15, suggest that PEA-15 expression is associated with a particular metabolic status of cells challenged with potentially apoptotic and/or proliferative signals.

SCA2 is not a major locus for ADCA type I in French families.

Autosomal dominant cerebellar ataxias (ADCA) of type I, a group of clinically heterogeneous neurodegenerative disorders, are known to be genetically heterogeneous since a second locus for ADCA type I (SCA2) has been identified on the long arm of chromosome 12. Linkage analysis was performed in 7 French ADCA type I families in order to estimate its frequency. We analysed 121 individuals, 39 of whom were affected. In 6 families, the SCA2 candidate interval, spanning 12.8 cM, was excluded by bi- and multipoint analysis. In one family (SAL-315), however, the maximal positive lod score reached 2.03 at the D12S79 locus. A posterior probability of 94% in favor of linkage to SCA2 was calculated by homogeneity analysis. The clinical profile of this family was similar to that of previously described SCA1 and non-SCA1 families, except that dementia was observed in 2 out of 6 patients. This may be a clinical idiosyncrasy in this family and was insufficient for a genotype-phenotype correlation.

Cyclic-amp dependent protein kinase in mouse striatal neurones and astrocytes in primary culture: development, subcellular distribution and stimulation of endogenous phosphorylation.

The cAMP-dependent protein kinase (cAMPdPK) activity and the endogenous cAMP-dependent phosphorylation of protein have been studied on pure populations of striatal neurones or astrocytes in primary culture originating from embryonic mouse brain. The appearance of cAMPdPK in cultured striatal neurones was rapid and paralleled neuritic outgrowth and cell maturation. Its highest value, reached between day 6 and 9 in culture, was comparable to that found in adult tissue. In cultured neurones as in adult striatum, cAMPdPK was found both in membrane and in cytoplasmic fractions. In astrocytes, cAMPdPK activity was low and only detectable in the cytoplasm. The presence of several cAMP-regulated phosphoproteins could be demonstrated in cultured cells, some of which being neurone-specific or astrocyte-specific. The phosphorylation of these striatal proteins was either enhanced, or, surprisingly, inhibited in the presence of cAMP. This study indicates that primary cultures of nerve cells provide valuable preparations for analysing protein phosphorylation processes induced by neurotransmitters.

Biogenic amine-sensitive adenylate cyclases in primary culture of neuronal or glial cells from mesencephalon.

Primary cultures of virtually pure mesencephalic neurons (5 days) or glials (4 weeks) from 14-day-old mouse embryo were obtained using appropriate medium. Membranes prepared from neuronal cells contained mainly serotonin and beta 1-adrenergic-sensitive adenylate cyclases. However, a low but significant classical dopamine-sensitive adenylate cyclase activity (D1 receptor) was detected. Contrasting with the data obtained from a previous study on striatal neurons no adenosine-sensitive adenylate cyclase was found on mesencephalic neurons. Study on the additive effects of the 3 biogenic amines-sensitive adenylate cyclases indicated that: all neuronal cells having dopamine receptors possess beta 1-adrenergic receptors (no additivity); beta 1-adrenergic and serotonin receptors on the one hand, and dopamine and serotonin receptors on the other hand, were coupled with independent adenylate cyclase systems localized either on two different domains of the same cell or on different cells (complete additivity). Membranes prepared from primary mesencephalic cultures of glial cells contained a mixture of beta 1- and beta 2-adrenergic receptor subtypes coupled with an adenylate cyclase (70% and 30%, respectively). No dopamine- or serotonin-sensitive adenylate cyclase was detected on mesencephalic glial cells.

Two simian virus 40 (SV40)-transformed cell lines from the mouse striatum and mesencephalon presenting astrocytic characters. I. Immunological and pharmacological properties.

Dissociate cultures were initiated from embryonic rostral mesencephalic and striatal tissues dissected from the mouse brain and previously incubated with a simian virus 40 (SV40) suspension. After several weeks in culture foci of fastly dividing cells were resuspended and cloned by successive dilutions. Several clones expressing the SV40 nuclear T antigen were obtained by these procedures and two of them, one mesencephalic (F7-Mes) and one striatal (F12-Str) were screened for the expression of glial or neuronal characters. Both clones possess adenylate cyclase-linked beta 2-adrenergic receptors. They also take up and synthesize gamma-aminobutyric acid (GABA) in amounts compatible with a glial origin. As is the case for astrocytes, the uptake of GABA is inhibited by beta-alanine and rather insensitive to the presence of diaminobutyric acid (DABA), a specific inhibitor of the neuronal GABA carrier. The most convincing evidence that F7-Mes and F12-Str belong to the astrocytic lineage comes from the fact that the two cell lines synthesize glial fibrillary acidic protein (GFAP) as demonstrated by immunofluorescence and immunoblotting. In an accompanying paper we also show that these lines behave like astrocytes when considered from the point of view of neuroglial interactions.

Dopamine-induced homologous and heterologous desensitizations of adenylate cyclase-coupled receptors on striatal neurons.

Striatal neurons in primary culture express a wide variety of adenylate cyclase-coupled receptors, and particularly dopamine (DA) D1 receptors, which are known to induce an increase in cyclic AMP production. To study desensitization of those receptor-mediated responses, neurons were incubated in the presence of saturating concentrations of DA for various times. We observed a rapid desensitization of the D1 response after 15 min, which reached a maximum after 18 h. This effect is reversible since incubation of treated neurons in fresh medium led to a complete recovery of the dopamine response within 2 days. In addition, a brief treatment with DA resulted in heterologous desensitization of beta-adrenoceptors and 5-HT and vasoactive intestinal peptide (VIP) receptors coupled to adenylate cyclase on striatal neurons. Similar to what was observed for homologous desensitization, these effects are obtained within the first 15 min of exposure to DA; however, they are short-lasting, even in the persistent presence of DA.

Somatostatin receptors on cortical neurones and adenohypophysis: comparison between specific binding and adenylate cyclase inhibition.

Primary cultures of mouse embryonic neurones from the cerebral cortex and rat pituitary membranes were used to identify and characterize further the somatostatin receptors coupled to an adenylate cyclase and to compare these receptors with specific binding sites for a non-reducible somatostatin analog. 125I-CGP 23996 on both tissues. 125I-CGP 23996 bound specifically to a single population of sites on cortical neurones and pituitary membranes, with a high affinity (Kd = 2.76 and 1.95 nM respectively). The rank order of potency of somatostatin-(1-14) and some analogs (somatostatin-28, [D-Trp8,D-Cys14]somatostatin-(1-14), native CGP) to displace 125I-CGP 23996 from its binding sites was similar on both tissues. Furthermore this rank order was also found identical for the inhibition of adenylate cyclase activity on cortical neuronal and pituitary membranes. Finally a good correlation was found between the order of potencies of somatostatin analogs evaluated from binding experiments and adenylate cyclase assays, suggesting the presence of the same receptor observed under two different affinity states. According to the classification of somatostatin receptors by Tran and his colleagues (1985) these results support the hypothesis that SSA is the somatostatin receptor coupled with an adenylate cyclase.

[Does the ataxo-choreic form of DRPLA exist in Europe? Search of mutation in 120 families]. / La forme ataxo-choréique de DRPLA existe-t-elle en Europe? Recherche de la mutation dans 120 familles.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by a degeneration of cerebellar and pallidal efferents, more frequent in Japan. Isolated cases are also encountered. Patients present with variable combination of signs including myoclonus, ataxia, epilepsy, choreoathetosis and dementia, with onset from childhood to the seventh decade. Clinically, DRPLA may be undistinguishable from other genetic disorders, in particular Huntington's disease or the spinocerebellar ataxias. The genetic basis of the inherited form of DRPLA is an expansion to more than 49 repeats of an unstable trinucleotide (CAG) in the DRPLA gene on the short arm of chromosome 12. We determined the frequency of this mutation in patients with the DRPLA phenotype. One hundred and seventeen patients with cerebellar ataxia, from 94 families and 23 isolated cases, as well as 3 patients from families with undiagnosed autosomal dominant neurodegenerative disorders were investigated for the presence of the expanded sequence. None of the patients carried this mutation. This finding suggests that DRPLA is rare in the French population. The search for the DRPLA mutation is justified in patients with the DRPLA phenotype, however, since genetic counselling is often requested and neither clinical, nor neuropathological examinations permit a definite diagnosis of the underlying disease.