Hervé Chneiweiss

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.

Vasoactive Intestinal Polypeptide Receptors Linked to an Adenylate Cyclase, and Their Relationship with Biogenic Amine- and Somatostatin-Sensitive Adenylate Cyclases on Central Neuronal and Glial Cells in Primary Cultures

Abstract: The presence of vasoactive intestinal polypeptide (VIP) receptors coupled to an adenylate cyclase was demonstrated on membranes of neurons or glial cells grown in primary cultures originating from the cerebral cortex, striatum, and mesencephalon of mouse embryos. A biphasic pattern of activation was observed in all these cell types, involving distinct high‐ and low‐apparent‐affinity mechanisms. The absence of additive effects of VIP and 3,4‐dihydroxyphenylethylamine (DA, dopamine), isoproterenol (ISO), and 5‐hydroxytryptamine (5‐HT, serotonin) suggests that the peptide receptors are colocated with each of the corresponding amine receptors on neuronal membranes of the three structures studied. The nonadditivity between the VIP‐ and ISO‐induced responses on cortical and striatal glial membranes reveals as well a colocation of VIP and β‐adrenergic‐sensitive adenylate cyclases on the same cells. A subpopulation of mesencephalic glia could possess only one of the two types of receptors, as a partial additivity of the VIP and ISO responses was seen. In addition, VIP modified the characteristics of the somatostatin inhibitory effect on adenylate cyclase activity of neuronal membranes from the cerebral cortex and striatum but not from those of the mesencephalon. On striatal and mesencephalic glial membranes the somatostatin inhibitory effect was observed only in the presence of VIP. However, as previously seen with ISO, the presence of VIP did not allow the appearance of a somatostatin inhibitory response on cortical glial membranes. This suggests that cortical glia are devoid of somatostatin receptors.

Cellular Expression, Developmental Regulation, and Phylogenic Conservation of PEA-15, the Astrocytic Major Phosphoprotein and Protein Kinase C Substrate

Abstract: PEA‐15 has recently been identified as a major phosphoprotein in astrocytes and an endogenous substrate for protein kinase C. This 15‐kDa protein exists under three molecular forms, an unphosphorylated form, N, and two phosphorylated forms, Pa and Pb. Ȧntisera were raised against synthetic peptides corresponding to the internal sequences of the mouse protein containing the two specific phosphorylation sites and affinity‐purified antibodies were used for immunoblotting. PEA‐15 was found mainly in the cytosol, but its protein kinase C‐phosphorylated form, Pb, was also detectable in association with the membrane and remained with the fraction that contains stabilized microtubules. Abundant in astrocytes, particularly in the hippocampus, PEA‐15 was also detected in all cultured brain cell types examined, indicating a more ubiquitous distribution of the protein, further demonstrated by its detection in the eye and in the lung. Parallel to the increase in expression levels, phosphorylation of PEA‐15 also increased during development. This paralleled results obtained in primary cultures, where PEA‐15 levels increase with cell maturation. Finally, physiological importance of PEA‐15 phosphorylation was illustrated by immunoreactivity observed in brain homogenates of different mammals, birds, amphibians, and fish.

Modulation by monoamines of somatostatin-sensitive adenylate cyclase on neuronal and glial cells from the mouse brain in primary cultures.

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin‐inhibitory effect on neuronal crude membranes was still observed in the presence of (—)‐isoproterenol, 3,4‐dihydroxyphenylethylamine (dopamine, DA), or 5‐hydroxytryptamine (5‐HT, serotonin) used at a concentration (10−5M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin‐inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5‐HT did not modify the somatostatin‐inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin‐inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (—)‐isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.

The multifunctional protein PEA-15 is involved in the control of apoptosis and cell cycle in astrocytes.

PEA-15 is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes [Araujo et al., J Biol Chem 1993;268(8):5911-20], and subsequently shown to be widely expressed in different tissues and highly conserved among mammals [Estelles et al., J Biol Chem 1996;271(25):14800-6; Danziger et al., J Neurochem 1995;64(3):1016-25]. It is composed of a N-terminal death effector domain and a C-terminal tail of irregular structure. PEA-15 is regulated by multiple calcium-dependent phosphorylation pathways that account for its different forms: a non-phosphorylated form in equilibrium with a mono and a biphosphorylated variety. This already suggested that PEA-15 may play a major role in signal integration. Accordingly, it has been demonstrated to modulate signaling pathways that control apoptosis and cell proliferation. In particular, PEA-15 diverts astrocytes from TNFalpha-triggered apoptosis and regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been modelized and recently determined using NMR spectroscopy, and may help to understand the various functions played by the protein through its molecular interactions.

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.

Stathmin Phosphorylation Is Regulated in Striatal Neurons by Vasoactive Intestinal Peptide and Monoamines via Multiple Intracellular Pathways

Abstract: Stathmin is a ubiquitous soluble protein whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell proliferation, differentiation, and functions by extracellular effectors. It is present in the various tissues and cell types as at least two distinct isoforms in their unphosphorylated (Mr∼ 19,000; pI ∼ 6.2–6.0) and increasingly phosphorylated forms. Stathmin is particularly abundant in brain, mostly because of its high concentration in neurons, where the protein is a major phosphorylation substrate. In intact striatal neurons grown in primary culture, the cyclic AMP–increasing drug forskolin and the protein kinase. C–activating agent 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) induced a potent phosphorylation of stathmin. Their actions were at least partially additive, appearing actually most likely “sequential” on various phosphorylated states of stathmin. Vasoactive intestinal peptide (VIP) reproduced the forskolin‐like stimulation but stimulated also other, TPA, and/or Ca2+‐like protein phosphorylations. These actions of VIP were already maximal after 5 min and were long lasting, still important after 2 h. In addition, concentrations as low as 1 nM were enough to obtain a significant effect, on both cyclic AMP‐dependent and independent phosphorylations. Dopamine and the β‐adrenergic agonist isoproterenol were also able to stimulate stathmin phosphorylation, but only with a forskolin‐like pattern. Their actions were not additive to those of VIP, confirming previous results on the colocalization of both dopamine D1 and nor‐adrenaline β1 receptors with VIP receptors on striatal neurons. In conclusion, our results show that VIP regulates the functions and differentiation of embryonic striatal neurons through multiple intracellular pathways and further substantiates the role of stathmin as a cytoplasmic relay integrating multiple second messenger signals.

p38/SAPK2 controls gap junction closure in astrocytes.

Astrocyte gap junction communication (GJC) is thought to contribute to death signal propagation following central nervous system injury, noteworthy in some ischemia/anoxia models. The inhibition of p38/stress‐activated protein kinase 2 (p38/SAPK2) by a pyrimidyl imidazole derivative has been reported to reduce the extent of the lesion area after cerebral ischemia. Therefore, interleukin‐1β (IL‐1β), which contributes to stroke‐induced brain injury and activates p38/SAPK2, and hyperosmolarity induced by sorbitol, a potent stimulus of p38/SAPK2 in non‐neuronal cells, were used to investigate a possible involvement of p38/SAPK2 in GJC modulation in mouse cultured astrocytes. Both stimuli inhibited dye coupling within minutes. The IL‐1β effect was transient, while that of sorbitol lasted up to 90 min. Both stimuli induced a rapid p38/SAPK2 activation, the kinetic of which matched that of induction of dye coupling inhibition. Immunocytochemical studies showed that IL‐1β and sorbitol induced a p38/SAPK2 translocation from the nucleus to the cytoplasm. The pharmacological agent SB203580 specifically blocked p38/SAPK2 activation, cytoplasmic translocation and reversed the IL‐1β and sorbitol‐induced inhibition of GJC. Further characterization of the p38/SAPK2 mode of action on GJC, performed with sorbitol, revealed an increased phosphorylation of protein kinase C (PKC) substrates abolished by both PKC inhibitors and SB203580. Expression and serine phosphorylation of connexin 43, the main component of astrocyte gap junctions, were unchanged, suggesting the existence of additional intracellular signaling mechanisms modulating the channel gating. Altogether, these results demonstrate that p38/SAPK2 is a central mediator of IL‐1β and sorbitol inhibitory actions on GJC and establish PKC among the distal effectors of p38/SAPK2.

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.

PEA‐15 Modulates TNFα Intracellular Signaling in Astrocytes

Abstract: PEA‐15 is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes and subsequently shown to be widely expressed in different tissues and highly conserved among mammals. It is composed of an N‐terminal death effector domain (DED) and a C‐terminal tail of irregular structure. PEA‐15 is regulated by multiple calcium‐dependent phosphorylation pathways. PEA‐15 is ideally positioned to play a major role in signal integration. Accordingly, it has been demonstrated that PEA‐15 diverts astrocytes from TNFα‐triggered apoptosis and regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. Expression of PEA‐15 directs TNFα outcomes toward survival, whereas its absence allows the development of the cytokine‐induced cell death.