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

Neuroscience 126 (2004) 263–275

THE EXPRESSION OF PEA-15 (PHOSPHOPROTEIN ENRICHED IN ASTROCYTES OF 15 kDa) DEFINES SUBPOPULATIONS OF ASTROCYTES AND NEURONS THROUGHOUT THE ADULT MOUSE BRAIN A. SHARIF,a F. RENAULT,a F. BEUVON,b R. CASTELLANOS,c B. CANTON,a L. BARBEITO,c M. P. JUNIERa1 AND H. CHNEIWEISSa1*

PEA-15 expression is associated with a particular metabolic status of cells challenged with potentially apoptotic and/or proliferative signals. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

a INSERM U114, Chaire de Neuropharmacologie, Colle`ge de France, 11 Place M. Berthelot, 75231 Paris, Cedex 05, France

Key words: PEA-15, astrocytes, neurons, neuroprogenitors, apoptosis, cell cycle.

b

Laboratoire d’Anatomopathologie, Hoˆpital Sainte-Anne, 75014 Paris, France c

Departamento de Neurobiologı´a Celular y Molecular, Instituto de Investigaciones Biolo´gicas Clemente Estable, Montevideo, Uruguay

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is a small protein (15 kDa) that was first identified as an abundant phosphoprotein in brain astrocytes (Danziger et al., 1995). It has subsequently been shown to be widely expressed in different tissues and highly conserved among mammals (Danziger et al., 1995; Estelles et al., 1996; Ramos et al., 2000). Several studies performed with primary cultures of astrocytes and various cell lines have demonstrated that PEA-15 regulates multiple cellular functions through its interaction with components of major intra-cellular transduction pathways. PEA-15 is composed of an N-terminal death effector domain (DED) and a Cterminal tail of irregular structure. We previously demonstrated that PEA-15 may bind other DED containing molecules such as Fas associated death domain (FADD) and caspase-8, resulting in a protection of astrocytes from tumor necrosis factor (TNF) ␣-triggered apoptosis (Estelles et al., 1999; Kitsberg et al., 1999). PEA-15 also opposes Fas-induced apoptosis in fibroblasts and glioma cell lines (Condorelli et al., 1999). DED-containing proteins are involved in other cellular signaling events besides the regulation of apoptosis (Tibbetts et al., 2003). Accordingly, PEA-15 activates the extra-cellular signal receptor-activated kinases (ERK1/2), members of the mitogen activated protein (MAP) kinase family (Ramos et al., 2000; Formstecher et al., 2001). It binds ERK in the nucleus and exports it to the cytosol, preventing the entrance into the cell cycle that depends from a sustained phospho-ERK nuclear accumulation (Brunet et al., 1999; Formstecher et al., 2001). PEA-15 has also been recently shown to interact with Akt and p90 ribosomal S6 kinase isozyme (RSK2), two key components of the phosphoinositide 3-kinase (PI3K) and ERK transduction pathways, the activations of which are known to be central to the modulation of cell survival (Trencia et al., 2003; Vaidyanathan and Ramos, 2003). In addition to these well-characterized effects, PEA-15 has also been shown to bind to and modulate the expression of phospholipase D (Zhang et al., 2000), and to increase the cells’ resistance to insulin in type II diabetes (Condorelli et al., 1998). PEA-15 thus functions as a fine

Abstract—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 antiproliferative 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 ⴚ/ⴚ 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 1

These authors equally participated to the supervision of this work. *Corresponding author. Tel: ⫹33-1-4427-1219; fax: ⫹33-1-4427-1260. E-mail address: [email protected] (H. Chneiweiss). Abbreviations: ALS, amyotrophic lateral sclerosis; DED, death effector domain; ERK, extracellular signal-regulated kinase; FADD, Fas associated death domain; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; MAP kinase, mitogen activated protein kinase; NGS, normal goat serum; PI3K, phosphoinositide 3-kinase; PBS, Dulbecco’s phosphate buffer saline; PEA-15, phosphoprotein enriched in astrocytes of 15 kDa; RSK2, p90 ribosomal S6 kinase isozyme; SOD, superoxyde dismutase; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.02.039

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tuner of mitogenic and trophic signaling in astrocytes, as well as in non-nervous cell types. To gain insights into PEA-15 functions in vivo, we conducted a thorough exploration of PEA-15 cellular sources in the normal adult brain. We show that normal astrocytes express PEA-15 in all areas of the brain. PEA-15 positive astrocytes represent only a subpopulation in each region examined, revealing a new heterogeneity among these cells. In addition, numerous labeled neurons were also observed. Furthermore, analysis of PEA-15 expression in a model of motor neuron degeneration accompanied with a robust astrogliosis (Gurney, 1994; Levine et al., 1999) revealed a marked upregulation of PEA-15 in reactive astrocytes.

EXPERIMENTAL PROCEDURES Production of the anti-PEA-15 antibody The anti-PEA-15 polyclonal antibody was raised in rabbits against the KLH-conjugated peptide SEEITTGSAWFSFLESHNK, dissolved in Freund’s complete adjuvant. This 19 amino acid sequence is located in the DED domain of the protein and does not encompass the serines submitted to phosphorylation (Araujo et al., 1993; Estelles et al., 1996; Kubes et al., 1998). Three subsequent injections of conjugate in Freund’s incomplete adjuvant were performed at 2 week intervals. Sera were tested for reactivity with the peptide by using a direct enzyme-linked immunosorbent assay. This polyclonal antibody was named Ab-7.

Animals All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Institut de Biologie du College de France. A minimal number of animals was used and all procedures regularly adopted to minimize their suffering. Construction of the PEA-15 ⫺/⫺ mice has been previously described (Kitsberg et al., 1999). The mice were bred in an airconditioned room with free access to water and food, and were separated and housed by sex 3 weeks after birth. Five PEA-15 ⫹/⫹ and five PEA-15 ⫺/⫺ adult mice (3–5 month-old) of both sexes were used for the immunohistochemical assays. Transgenic mice for G93A human SOD1 strain B6SJL-TgN(SOD1G93A)1Gur (Gurney, 1994) mice (B6.129S4-Ngfrtm1Jae, Stock Nr 002213) were purchased from Jackson Laboratory (Bar Harbor, ME, USA).

Western blot analysis Mice were overdosed with pentobarbital (600 mg/kg), and the brains rapidly dissected. The whole brains were homogenized by 15 strokes using a Teflon pestle and a Potter glass homogenizer in a lysis buffer containing 10 mM Tris, 50 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 50 mM NaF, 5 ␮M ZnCl2, 100 ␮M Na3VO4, 1 mM DTT (1 ml lysis buffer per brain). The lysates were clarified by centrifugation (15 min; 15,000⫻g). The protein levels were determined using the BCA assay (Pierce, Rockford, IL, USA). Sixty microgram lysates were resolved on a 10 –20% Ready gel (BioRad, Hercules, USA). The proteins were transferred onto Hybond-C Extra nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway NJ, USA) with a BioRad semi-dry blotting apparatus (15 V, 1 h). The membranes were stained with Ponceau Red (0.2% in 1% acetic acid) prior incubation for 1 h at room temperature in TNT buffer containing 5% non-fat dry milk (50 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween 20). They were then incubated overnight at 4 °C with the Ab-7 anti-PEA-15 antibody (1:5000) in TNT buffer containing 5% nonfat dry milk. Bound antibodies were detected with anti-rabbit IgG

coupled to horseradish peroxidase (1:2500; Amersham) and revealed by the enhanced chemiluminescence Amersham ECL detection system according to the manufacturer instructions. Membranes were then immunoblotted with a monoclonal mouse antiactin antibody (1:1000; Chemicon International, Temecula, CA, USA) as described above.

Cell lines and culture The green fluorescent protein, 3T3-GFP and 3T3-GFP–PEA-15 cell lines have been previously described (Kitsberg et al., 1999; Formstecher et al., 2001). Briefly, they were obtained by transfecting NIH 3T3 cells with the plasmids pEGFP-C1 (Clontech) and pEGFP-PEA-15 (Kitsberg et al., 1999), respectively. Two clones resistant to G418 were selected for each construct and cultured in DMEM (Roche) supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (5 IU/ml) and streptomycin (5 ␮g/ml). Expression of GFP and GFP-PEA-15 was checked by fluorescence of living cells and Western blot analysis with anti-GFP and antiPEA-15 antibodies. Primary cultures of astrocytes were prepared from embryonic day 16 PEA-15 ⫺/⫺ or PEA-15 ⫹/⫹ mouse cortices and striata following previously described procedures (Araujo et al., 1993).

Immunocytochemistry The cells were washed twice with PBS (Dulbecco’s phosphate buffer saline, without calcium chloride and magnesium chloride; Sigma), fixed with 4% paraformaldehyde in PBS (pH 7.5) for 15 min and washed twice with PBS containing 0.1 M glycine at room temperature. They were then incubated for 5 min in PBS containing 0.2% Triton X-100. Nonspecific sites were blocked with PBS containing 10% normal goat serum (NGS) for 1 h at room temperature. Cells were incubated overnight at 4 °C with the antiPEA-15 antibody diluted at a concentration of 1/5000 in PBS containing 1.5% NGS. After three washes in PBS, cells were incubated for 1 h at room temperature with CY3-conjugated antirabbit antibody (Amersham, France) diluted at a concentration of 1/500 in PBS⫹1.5% NGS. The nuclei were stained with Hoechst Dye according to manufacturer’s specifications (Hoechst). The coverslips were mounted under glass slides in Fluoromount medium (Southern Biotechnology) and examined on a confocal microscope (TCS SP2; Leica) or a fluorescent microscope (Eclipse E800; Nikon) with appropriate filters. For confocal analysis, the excitation wavelengths were 543 nm for Cy3, 488 nm for FITC, 633 nm for TOPRO, and the emission wavelengths were 570 nm for Cy3, 510 –525 nm for FITC and 647 nm for TOPRO. For fluorescent microscopy, we used custom made filters (Chroma): the excitation wavelengths were 440 – 480 nm for GFP, 530 – 545 nm for CY3 and 340 –380 for DAPI. The emission wavelengths were 535–550 nm for GFP, 620 – 660 nm for CY3 and 435– 485 nm for DAPI.

Immunohistochemistry Mice were overdosed with pentobarbital (600 mg/kg) and perfused with 0.1 M PBS (pH 7.4), followed by 4% paraformaldehyde in PBS. The dissected brains were post-fixed in 4% paraformaldehyde for 4 h at 4 °C, cryoprotected in 30% sucrose in PBS at 4 °C until the tissue sank, frozen in ⫺40 °C isopentane, and stored at ⫺80 °C. Cryostat sections (20 ␮m thickness) were cut in the frontal plane. Immunohistochemistry was performed on free-floating sections. Fifteen sections, from the olfactory bulbs to the posterior end of the cerebellum, were selected for the analysis. The spinal cords of the 120 day-old G93A SOD1 mice and of their age-matched controls were paraffinembedded after the post-fixation step. The blocks were sectioned at 5 ␮m thickness on a microtome. Cryostat sections were first incubated in 0.3% H2O2 in PBS for 15 min to quench endogenous peroxidase activity while spinal cords section were first deparaf-

A. Sharif et al. / Neuroscience 126 (2004) 263–275 finized prior to be incubated at ⫺20 °C in 0.3% H2O2 in methanol for 20 min. All tissues were then incubated in PBS containing 0.3% Triton X-100. The sections were immunostained with either one of the following antibodies: anti-PEA-15 Ab7 antibody (1:9000), monoclonal mouse anti-glial fibrillary acidic protein (GFAP; 1:500; ICN, France; or CY3-conjugated anti-GFAP; 1:400; Sigma), a marker of astrocytes, monoclonal mouse anti-NeuN (1:1000; Chemicon, France; Mullen et al., 1992), a marker of neurons, and monoclonal rat anti-Mac1 (anti-CD11b receptor; 1:100; PharMingen, France), a marker of microglial cells (Graeber et al., 1988; Rabchevsky et al., 1998). Prior to use for immunohistochemistry, the anti-PEA-15 antibody was incubated overnight at 4 °C under agitation with 20 ␮m thick brain sections of PEA-15 ⫺/⫺ mice (eight sections per 250 ␮l of antibody diluted to 1:9000 in PBS, 0.3%Triton, 0.1%BSA or 1:1000 in paraffin sections). The primary antibodies were applied overnight at room temperature or at 4 °C for the anti-PEA-15 antibody. The secondary antibodies used were a biotinylated anti-rabbit antibody (1:300; Vector Laboratories, France), an anti-mouse IgG1-FITC antibody (1:500; Southern Biotechnology, France), an anti-rat-FITC antibody (1:400; Southern Biotechnology) and an anti-rabbit-FITC antibody (the one used to reveal the anti-PEA-15 in the spinal cord dilution, manufacturer). PEA-15 immunohistochemical detection was achieved using either the avidin– biotin complex immunoperoxidase technique and the diaminobenzidine chromogen (Vector Laboratories, Burlingame, CA, USA) or the tyramide signal amplification system coupled to the fluorochrome cyanine three (1:400 in PBS, 10 min, at room temperature; Perkin-Elmer Life Sciences, Inc, USA). Hematoxylin (Mayer hematoxylin; Merck; undiluted, 5 s at room temperature) and DAPI or TOPRO (Molecular Probes; 1:2000 in PBS, 10 min, at room temperature) were used to visualize the cells nuclei after immunolabeling with the diaminobenzidine chromogen or fluorochromes, respectively. Immunofluorescence was observed with a fluorescent microscope (Eclipse E800; Nikon). Images were acquired on a Digital still camera (DXM 1200; Nikon) using the Lucia software (Laboratory Imaging, Ltd). For confocal immunofluorescence, slides were observed using a Leica TCS SP2 confocal microscope. Three lasers were used depending on the fluorochrome. The excitation wavelengths were 543 nm for Cy3, 488 nm for FITC, 633 nm for TOPRO, and the emission wavelengths were 570 nm for Cy3, 510 –525 nm for FITC and 647 nm for TOPRO. Tissue sections were observed with the 16, 40 and 63Å objectives, with the 0.50, 1.25 and 1.32 numerical aperture lenses, respectively (Leica, Germany). The pinhole was set on the airy spot and the images were acquired in a sequential scan mode. The resolution on x–y was from 0.5–2 ␮m for z. The images were prepared for printing using Adobe Photoshop software (Adobe Systems, San Jose, CA, USA).

RESULTS Characterization of the anti-PEA-15 antibody Ab-7 Immunoblotting assays of protein lysates prepared from adult PEA-15 ⫹/⫹ mice brains revealed a unique 15 kDa immunoreactive band. No signal was detected on protein lysates prepared from adult PEA-15 ⫺/⫺ mice (Fig. 1A). Further analysis in 2D-gels indicated that Ab7 recognizes the phosphorylated as well as the unphosphorylated forms of the protein (data not shown). Indeed, the antigen used to generate Ab-7 does not encompass the serines 104 and 116, which are submitted to phosphorylation. Further evaluation of the specificity of the Ab-7 antibody was carried out using 3T3 cells stably transfected either with the GFPPEA-15, or with the GFP alone constructs. In GFP-transfected 3T3 cells, a weak immunoreactive signal,

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mainly localized in the cytoplasm, was observed (Fig. 1B). The weakness of the signal appears to be linked to the low levels of endogenous PEA-15 in 3T3 cells (unpublished observations). The PEA-15-immunoreactive signal did not overlap with the diffuse green fluorescent GFP signal (compare Fig. 1B and 1C). In contrast, in GFP-PEA-15transfected 3T3 cells, Ab-7 yielded an immunoreactive signal that was strictly co-localized with the signal emitted by the GFP-PEA-15 fusion protein (compare Fig. 1D and 1E). Furthermore, in PEA-15 ⫹/⫹ primary cultures of astrocytes, PEA-15 immunoreactivity was observed predominantly in the cytoplasm (Fig. 1F) while no immunolabeling was detected in PEA-15 ⫺/⫺ astrocytes (Fig. 1G). Finally, immunohistochemical labeling of PEA-15 ⫺/⫺ mouse brains sections did not yield any immunoreactive signal while PEA-15 immunoreactive cells were observed in various regions of PEA-15 ⫹/⫹ mouse brains (compare Fig. 2A and B, and Fig. 4A and B). PEA-15-immunohistochemistry reveals a widespread expression of the protein throughout the normal adult brain PEA-15 immunoreactivity was examined in the brain of wild-type mice as compared with PEA-15 ⫺/⫺ mice. Numerous PEA-15-immunoreactive astrocytes and neurons were detected in all the CNS regions examined, throughout the rostro-caudal extent of the mouse adult brain (Table 1). No overt difference in the PEA-15 immunoreactivity profile was observed between both sexes. Astrocyte-like cells were characterized by a small-sized nucleus and soma, and a stellate shape after hematoxylin counterstaining of immunolabeled brain sections (see insert in Fig. 2D for example). Their identity was further confirmed using double PEA-15 and GFAP immunofluorescent stainings (Fig. 3D, and Fig. 6D–I). PEA15-immunoreactive astrocytes were observed in the gray matter of most of the brain areas examined (Table 1). Some PEA-15-immunoreactive astrocytes were also detected in white matter tracts, such as the corpus callosum, the cerebellar peduncles, and the fornix (Table 1; Fig. 2E and F). Aside astrocytes, PEA-15 labeling also revealed numerous positive neurons scattered throughout the brain. The neurons were identified by their large soma and nucleus (Fig. 4) and further confirmed to be neurons using double immunofluorescent stainings of PEA-15 and NeuN (insets in Fig. 4A and F). Only part of the neuronal population of a given cerebral area was immunoreactive for PEA-15, as revealed by double PEA-15 and NeuN immunofluorescent stainings (illustrated in insets in Fig. 4A and F). Such an immunoreactive pattern included the areas presenting a high density of PEA15-immunoreactive neurons such as the dentate gyrus of the hippocampus (Fig. 3F and Fig. 4D) or the piriform cortex and the amygdala (Fig. 4A and C). In all neurons, PEA-15-immunoreactive signal was observed in the soma cytoplasm (Fig. 4A, and 4D–F). Labeling of their neurites varied according to the brain area considered. For instance, in the median raphe only the proximal part of the neurites was occasionally labeled (Fig. 4F). In contrast, the neuritic arborization in the

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Fig. 1. Characterization of the anti-PEA-15 antibody. The anti-PEA-15 antibody recognizes in a specific manner the PEA-15 protein in immunoblotting assays (A) Protein lysates from adult PEA-15 ⫹/⫹ (lane 1) or PEA-15 ⫺/⫺ (lane 2) mice brains were Western blotted with the anti-PEA-15 antibody. An immunoreactive band was only detected on PEA-15 ⫹/⫹ protein lysates. (B, C) NIH 3T3 cells stably transfected with the GFP construct were stained with the anti-PEA-15 antibody. The weak red PEA-15 immunocytochemical signal localized in the cytoplasm (B) did not overlap with the diffuse green GFP signal (C). (D, E) NIH 3T3 cells stably transfected with the GFP-PEA-15 construct were stained with the anti-PEA-15 antibody and yielded to a red immunoreactive signal (D) that was strictly colocalized with the green GFP signal (E). (F, G) Primary cultures of cortico-striatal astrocytes were immunostained with the anti-PEA-15 antibody. The red PEA-15 immunocytochemical signal was mainly localized in the cytoplasm of PEA-15 ⫹/⫹ astrocytes (F), while no signal was detected in PEA-15 ⫺/⫺ astrocytes (G). Scale bar⫽20 ␮m. All images were obtained on a confocal microscope.

piriform cortex and amygdala, as well as the dendritic and axonal arborization of dentate gyrus neurons were heavily labeled (Fig. 3A, Fig. 4C and D). The majority of the cerebral regions presented both an astrocytic and a neuronal immunoreactivity (Table 1) such as in the hippocampus. This CNS area exhibited PEA-15 immunoreactive astrocytes in the CA1 and CA3 areas (Fig. 3A–E), and PEA-15-immunoreactive granular neurons in the dentate gyrus (Fig. 3F and Fig. 4D) as well as a few pyramidal neurons (not shown). A few

brain regions presented an exclusive astrocytic or neuronal pattern of PEA-15 immunoreactivity. In the olfactory bulbs and the striatum, PEA-15 immunolabeling was only observed in astrocytes (Fig. 2A and C). Conversely, PEA-15 immunoreactivity was only detected in neurons in the anterior cortex, the ventro-median area of the hypothalamus, the raphe or the periaqueductal gray (Table 1; Fig. 4E and F). No PEA-15 immunoreactivity was detected in endothelial cells (data not shown). Double immunofluorescent

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Fig. 2. PEA-15 expression reveals subpopulations of astrocytes throughout the adult brain. Brain sections of PEA-15 ⫹/⫹ (A, C–F) and PEA-15 ⫺/⫺ (B) mice were immunostained with the anti-PEA-15 antibody using the diaminobenzidine chromogen (brown) followed by hematoxylin counterstaining of the nuclei (blue). No immunoreactive signal was detected using PEA-15 ⫺/⫺ mice brain sections (B). PEA-15 immunoreactive signals were observed in all brain regions in the gray matter, such as the olfactory bulbs (A), the striatum (C) and the ventral part of the thalamus (D), as well as in white matter tracts, such as the corpus callosum (E) and the cerebellar peduncles (F). Arrows point to astrocyte-like cells. aci, anterior commissure, intrabulbar part; cp, cerebellar peduncles; cx, cortex; ec, external capsule; EPl, external plexiform layer of the olfactory bulb; GrO, granular cell layer of the olfactory bulb; st, striatum. Scale bar⫽50 ␮m (A–D and F), 20 ␮m (E and insets in C, D, F).

stainings using PEA-15 and the microglial marker Mac1 did not reveal any PEA-15 immunoreactive signal in microglia in the different regions examined i.e. the striatum, the hippocampus, the thalamus, and the cerebellum (data not shown). PEA-15 immunoreactivity was also observed in the subventricular zone (SVZ, Fig. 5A), an area of neurogenesis in the adult CNS. PEA-15-immunoreactive cells were

located in the most rostral and dorsal area of the SVZ, along the lateral wall of the lateral ventricle and at its dorsal tip, where subependymal progenitor cells are located (Tramontin et al., 2003). In addition, PEA-15 immunoreactive cells were also observed in the rostral migratory stream (RMS, Fig. 5B). In the SVZ and the RMS, PEA-15immunoreactive cells appeared essentially arranged as chains.

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Fig. 3. Confocal microphotographs of PEA-15 immunoreactive astrocytes and neurons in the hippocampus. Double immunofluorescent stainings were performed using the anti-PEA-15 (red) and GFAP (green) antibodies on brain sections of PEA-15 ⫹/⫹ mice. (A–C) Overview of the CA1 and dentate gyrus (DG) areas of the hippocampus. (A) PEA-15-immunoreactive astrocytes in CA1 and granular neurons in the DG. Note the dense immunoreactive signal of the dendritic and axonal arborizations of the granular neurons. (B) GFAP immunostaining. (C) overlay of (A) and (B). (D) Colocalization of PEA-15 and GFAP immunoreactive signals in the CA1 area of the hippocampus. All GFAP-immunoreactive astrocytes presented a PEA-15 immunoreactive signal in this area. (E) Colocalization of PEA-15 and GFAP immunoreactive signals in the CA3 area of the hippocampus. Only part of the GFAP-immunoreactive astrocytes presented a PEA-15 immunoreactive signal in this area. Arrowheads point to GFAP-immunoreactive astrocytes devoid of PEA-15 immunoreactive signal. Arrows point to astrocytes immunoreactive for both GFAP and PEA-15. (F) Immunohistochemical detection of GFAP and PEA-15 in the DG area. No PEA-15 immunopositive astrocytes were detected in this region. Note the PEA-15 immunoreactive granular neurons in the DG. The arrow points to a PEA-15-immunoreactive granular neuron and the arrowhead to a granular neuron devoid of PEA-15 immunoreactivity. Scale bars⫽160 ␮m in A–C, and 20 ␮m in D–F.

PEA-15 expression defines sub-populations of astrocytes in the normal adult brain Double immunofluorescent staining of brain sections with GFAP and PEA-15 antibodies showed that PEA-15 was mainly localized in the astrocytes cytoplasm, and extended to the processes (Fig. 6D–I). Likewise, in cultured astrocytes, PEA-15 appeared mainly localized in the cytoplasm of the cells, with a perinuclear reinforcement while a weak signal was detected in the nucleus (Fig. 6A). These observations are in accordance with results based on GFPPEA-15 transfected astrocytes showing that the nuclear export sequence of PEA-15 prevents its accumulation into the nucleus (Formstecher et al., 2001). Although PEA-15-immunoreactive astrocytes were observed in most structures of the adult brain, only part of them expressed PEA-15 in a given cerebral area, PEA-15immunoreactive astrocytes being scattered among PEA15-immunonegative astrocytes. This was in marked contrast with the in vitro observations. Double immunofluorescent labeling of cortico-striatal astrocytic cultures with PEA-15 and GFAP antibodies revealed that all astrocytes

present in the cultures exhibited a robust PEA-15-immunoreactive signal (Fig. 6A–C). The hippocampus showed a unique expression profile. In this structure, PEA-15 positive and negative astrocytes were spatially segregated. PEA-15-immunoreactive astrocytes were restricted to the CA1 and CA3 regions (Fig. 3A, D and E). No PEA-15-immunoreactive astrocytes were detected in the dentate gyrus area (Fig. 3F). In addition, double immunofluorescent labeling of GFAP and PEA-15 revealed that the vast majority of GFAP-immunolabeled cells of the CA1 area were also PEA-15 immunoreactive (Fig. 3D), while only part of the GFAP-immunolabeled astrocytes of the CA3 area exhibited a PEA-15 immunoreactive signal (Fig. 3E). Reactive astrocytes express PEA-15 in the G93ASOD1 mutant mouse To determine whether PEA-15 expression may be regulated in vivo, we analyzed a pathological situation challenging cell survival and cell cycle, namely astrogliosis. Astrogliosis is characterized by profound changes in astrocytes, which pass from quiescent to reactive morpholo-

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Fig. 4. PEA-15 expression reveals subpopulations of neurons throughout the adult brain. Brain sections of PEA-15 ⫹/⫹ (A, C–F) and PEA-15 ⫺/⫺ (B) mice were immunostained with the anti-PEA-15 antibody. (A, B, E and F) PEA-15 immunohistochemical staining using the diaminobenzidine chromogen (brown) followed by hematoxylin counterstaining (blue). (Insets in A and F) Double immunofluorescent stainings using the anti-PEA-15 antibody (red) and the neuronal specific marker NeuN (green). (C) Double immunofluorescent stainings of PEA-15 (red) and GFAP (green). (D) Immunofluorescent stainings of PEA-15 (red). (A and B) Numerous PEA-15-immunoreactive neurons were observed in the piriform cortex and the amygdala of ⫹/⫹ mice (arrows), while no immunoreactive signal was detected using PEA-15 ⫺/⫺ mice brain sections (B). Inset in A shows double PEA-15 and NeuN immunoreactive neurons (arrow) next to a NeuN-immunopositive neuron devoid of PEA-15 signal (arrowhead). (C) In the piriform cortex and the amygdala, PEA-15-immunoreactive neurons prevailed over rare PEA-15-immunoreactive astrocytes identified by their GFAP expression (arrowheads). Note that the immunoreactive signal is localized in both the neuronal soma and neurites. (D) PEA-15-immunoreactivity was detected in the soma of part of the granular neurons of the dentate gyrus as well as in the dendrites of the granule cells in the molecular layer (asterisk) and in their axons in the stratum lucidum (cross). (E) PEA-15-immunoreactive neurons (arrows) are observed in the ventro-median nucleus of the hypothalamus. (F) PEA-15-immunoreactive neurons in the median raphe (arrows). Inset shows double PEA-15 and NeuN immunoreactive neurons (arrow) next to NeuN immunopositive neurons lacking PEA-15 expression (arrowhead). Pir, piriform cortex; PLCo, posterolateral cortical amygdaloid nucleus. Scale bar⫽50 ␮m in A, B, E and F, 40 ␮m in C and D, 20 ␮m in insets in A, and F. Images in A, B, E and F were obtained on a light microscope, and images in C, D, and insets in A and F were obtained on a confocal microscope.

gies and metabolic profiles (Bignami and Dahl, 1974; Dahl and Bignami, 1974; Eng, 1988; Eddleston and Mucke,

1993; Ridet et al., 1997). This process is systematically associated with CNS trauma and neurodegenerative dis-

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Table 1. Summarized information on areas expressing PEA-15 in astrocytes and/or neurons of the adult mouse braina Areas Telencephalon Olfactory bulbs Neocortex Basal ganglia Striatum Nucleus accumbens Septum Amygdala Piriform cortex Hippocampus CA1–CA3 Dentate gyrus Bed nucleus of the stria terminalis Diencephalon Hypothalamus Thalamus Subthalamus Mesencephalon Superior and inferior colliculi Central gray Pontine nuclei Metencephalon Cerebellum Pons Periaqueductal gray Raphe Nucleus of the solitary tract Inferior olive Nucleus V Nucleus VII Nucleus XII Lateral lemniscus nucleus White matter Corpus callosum Fornix Anterior commissure Cerebellar pedoncules Pyramidal tract

Astrocytes

Neurons

⫹ ⫹

⫺ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫹ ⫹ ⫹ ⫹

⫹ ⫺ ⫹

⫹ ⫹ ⫹

⫹ ⫹ ⫺

⫹ ⫹ ⫹

⫹ ⫺ ⫹

⫹ ⫹ ⫹

⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

a Detection of immunoreactive cells in a given area is symbolized with a (⫹), and lack of detection with a (⫺).

eases such as amyotrophic lateral sclerosis (ALS; Norenberg, 1994; Newbery and Abbott, 2001; Wong et al., 1998). In ALS patients and in transgenic mice bearing mutated forms of human superoxyde dismutase, SOD1, that reproduce the pathology of a familial form of ALS, degenerating motor neurons are surrounded by reactive astrocytes (Hirano, 1996; Levine et al., 1999; Newbery and Abbott, 2001; Howland et al., 2002; Hensley et al., 2003). These astrocytes display the characteristic hallmarks of astrogliosis including hypertrophied astrocytic morphology with enlarged soma and thickened processes, enhanced expression of the GFAP gliofilament, and up-regulation of metabolic pathways associated with increased stress inputs (Chung et al., 2003; Feeney et al., 2003; Hensley et al., 2003). With regard to the antiapoptotic and anti-proliferative properties of PEA-15, we thus thought to explore an eventual regulation of PEA-15 expression by astrocytes in

the G93A mutant mice. We compared the labeling pattern of PEA-15 in the ventral horn of the lumbar spinal cord of 120 day-old wild-type mice, and of age-matched mutants that display overt symptoms of paralysis of the hind limbs. In the wild-type mice, a low PEA-15-immunoreactive signal was observed in some astrocytes (Fig. 7A) and neurons (Fig. 7C and E). On the opposite, a marked PEA-15 immunolabeling was detected in the G93A mutants in the reactive astrocytes that had developed throughout the spinal gray matter (Fig. 7F, G). Furthermore, a marked PEA-15 immunoreactive signal was observed in the motor neurons that, at this time, had survived the degenerative process (Fig. 7B).

DISCUSSION Up to this day, in vitro studies have led to consider PEA-15 as a protein enriched in cultured astrocytes where it acts as an inhibitor of apoptosis and cell proliferation. Here we confirm, using a novel anti-PEA-15 antibody suitable for immunohistochemistry, a widespread expression of the protein in the normal mouse brain in subpopulations of astrocytes. In addition, we show that neurons may also be cellular sources of PEA-15 in the mouse brain. Further in vivo analysis of PEA-15 regulation revealed that, in a mouse model of neurodegeneration accompanied with astrogliosis, reactive astrocytes up-regulate PEA-15 expression. The restricted expression of the protein to subpopulations of astrocytes and its up-regulation in pathological situations, combined with the known properties of PEA-15, raise the possibility that PEA-15 is synthesized by cells challenged with potentially apoptotic and/or proliferative signals. PEA-15 cellular sources correspond to astrocytes and neurons disseminated throughout the adult brain PEA-15 was first characterized as a highly phosphorylated protein enriched in astrocytes grown in primary cultures (Araujo et al., 1993). The initial immunoblotting assays of protein lysates derived from various brain areas of adult mice led to PEA-15 detection in all brain regions studied (Danziger et al., 1995). Accordingly, we observed PEA-15immunoreactive cells throughout the rostro-caudal extent of the murine brain. The study of Danziger et al. (1995) had shown a similar expression level in most of the brain structures with the exception of the hippocampus, which exhibited two-fold higher PEA-15 levels than all the other areas analyzed. Accordingly, the hippocampus presented also the highest apparent density of PEA-15-immunoreactive cells. Previous quantification of PEA-15 by immunoblotting assays in embryonic cultures, had shown that the levels of the PEA-15 protein in astrocytes were 20-fold higher than in oligodendrocytes and RBE4 cells, an immortalized endothelial cell line, and 10-fold higher than in neurons and microglia (Danziger et al., 1995). These data suggested that, at least in embryonic cells cultures, astrocytes were the main cellular source of PEA-15. The present study reveals that in the adult murine brain, both astrocytes and

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Fig. 5. PEA-15 expression was detected in an area of neurogenesis in the adult brain. Brain sections of PEA-15 ⫹/⫹ mice were immunostained with the anti-PEA-15 antibody. PEA-15 immunoreactive signal appears in brown and nuclei in blue. Arrows point to PEA-15 immunoreactive cells located in the subventricular zone, at the dorsal tip of the lateral ventricle (A) and in the rostral migratory stream (B). Most PEA-15 immunopositive cells detected in both of these regions were arranged as chains. cc, corpus callosum; LV, lateral ventricle; RMS, rostral migratory stream. Scale bar⫽10 ␮m.

neurons are cellular sources of PEA-15, the PEA-15immunoreactive neurons appearing grossly as numerous as the PEA-15-immunoreactive astrocytes. The pattern of PEA-15 expressing neurons did not overlap that of broad

functional networks. Likewise, in well-defined neuronal populations, PEA-15 immunoreactive profile differed from previously described phenotypes. Up to this day, no exploration of the role(s) sustained by PEA-15 in neurons has

Fig. 6. Intracellular localization of PEA-15 in astrocytes in vitro and in situ. Fluorescent microscopy (A–C) or confocal visualization (D–I) of PEA-15 (red) and GFAP (green) immunoreactivities. (A–C) Primary cultures of cortico-striatal astrocytes. Note that the PEA-15 immunoreactive signal is mainly localized in the cytoplasm. (D–F) A PEA-15 immunopositive astrocyte in the striatum of an adult mouse. (G–I) A PEA-15 immunopositive astrocyte of the corpus callosum of an adult mouse. In situ, PEA-15 is predominantly localized in the cytoplasm of astrocytes. C, F and I show the overlay of the red and green immunoreactive signals. Scale bar⫽20 ␮m in A–C, 8 ␮m in D–I.

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Fig. 7. PEA-15 expression in the lumbar spinal cord of 120-day-old G93A SOD1 transgenic mice displaying clinical and histological symptoms of the motor neurons degeneration as compared with a age-matched non-transgenic littermate. (A and B) Ventral horn of the lumbar spinal cord of a non-transgenic (A) and a G93A SOD1 transgenic (B) mouse immunostained with the anti-PEA-15 antibody using the diaminobenzidine chromogen (brown) followed by hematoxylin counterstaining of the nuclei (blue). (A) In the ventral horn of the lumbar spinal cord of the non-transgenic mouse, a low PEA-15-immunoreactive signal was observed in some astrocytes (arrows) and neurons. (B) In contrast, in the ventral horn of the lumbar spinal cord of the G93A SOD1 transgenic mouse, a marked PEA-15 immunolabeling was detected in the reactive astrocytes that had developed throughout the spinal gray matter (arrows) and in the motor neurons that, at this time, had survived the degenerative process (arrowheads). Scale bar⫽40 ␮m. (C–E) Double immunofluorescent stainings using the anti-PEA-15 (C, green) and GFAP (D, red) antibodies in the lumbar spinal cord of 120-day-old non-transgenic and G93A SOD transgenic mice, respectively. The overlay of the PEA-15 and GFAP staining is shown in E. Arrowheads in C and E point to PEA-15 immunoreactive neurons. Note that in the lumbar spinal cord of wild-type mice, GFAP-immunolabeled astrocytes are essentially detected in the white matter visible at the left extremity of the picture. (F–H) Double immunofluorescent staining of PEA-15 (F, green) and GFAP (G, red) in the lumbar spinal cord of a 120-day-old G93A SOD1 transgenic mouse. The overlay of F and G is shown in H. Note that in the transgenic mice, the gray matter of the lumbar spinal cord contains numerous astrocytes immunoreactive for both GFAP and PEA-15. Scale bar⫽100 ␮m.

been undertaken. The discovery of an in vivo neuronal expression of PEA-15, associated with the observation of an up-regulation of PEA-15-immunoreactive signal in the SOD1 mutant mouse degenerating motor neurons, stresses the necessity to address this issue. In contrast with the results that we previously obtained in vitro (Danziger et al., 1995), we did not observe microglia or endothelial cells expressing PEA-15 in the various regions examined. This discrepancy does not result from the use of a different antibody. Immunoblotting assays of protein lysates derived from cultured microglia and endothelial cells with the novel Ab-7 antibody yielded results similar to those previously published (Danziger et al., 1995). It rather may stem either from the culture conditions and/or the differentiation status of the cells. In addition to astrocytes and neurons, a few PEA-15-immunoreactive oligodendrocyte-like cells were observed (data not shown).

PEA-15 expression unveils subpopulations of astrocytes in the adult brain One of the most noticeable characteristics of the expression profile of PEA-15 in the adult murine brain is its presence in only a subset of astrocytes, regardless of the CNS area considered. It is now well established that astrocytes form a highly heterogeneous population at both the morphological and functional levels. The astrocytes subpopulations have so far been essentially related to their regional localization. From one region to another, the astrocytes morphology and their content in intermediate filaments vary (Bignami and Dahl, 1974; Eng, 1988; Nolte et al., 2001). They also exhibit different combinations of neurotransmitters receptors, amino acids transporters, ionic channels, growth factors, growth factor receptors, as well as different actions on neurons (Denis-Donini et al., 1984; Chneiweiss et al., 1985; Barres et al., 1990; Wilkin et al., 1990; Ma et al., 1994; Lehre et al., 1995; Muller and

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Kettenmann, 1995; Porter and McCarthy, 1996; Poopalasundaram et al., 2000). Such differences have also been uncovered within a given brain region, albeit at a lesser extent (McCarthy and Salm, 1991; Israel et al., 2003; Matthias et al., 2003; Reuss et al., 2003). For example, the coexistence of astrocytes containing low and high GFAP levels has been reported (Stichel et al., 1991; Walz and Lang, 1998). PEA-15-immunoreactivity was however detected in both types of astrocytes. It was visualized in striatal astrocytes, which exhibited a low GFAPimmunoreactive fluorescent signal, as well as in hippocampal astrocytes, which presented a high GFAPimmunoreactive signal. Another example is the hippocampus, into which several attempts have recently been done to correlate the astrocytes molecular phenotype to their electrophysiological activity. These studies have led to the identification of “passive” and “complex” subtypes according to their expression of voltage currents, GFAP, S100␤ and glutamate synthase. Although the “passive” astrocytes appear more abundant in the CA1 area and the “complex” astrocytes in CA3, both types may be found in either area and most authors consider these phenotypes as the two extremes of a spectrum reflecting the astrocytes plasticity (Walz, 2000). PEA-15 astrocytes appeared more spatially segregated than the complex and passive astrocytes, most astrocytes expressing PEA-15 in the CA1 area, only part of them in the CA3 area and none being detected in the dentate gyrus region. PEA-15 expression appears thus to reveal a novel sub-population of astrocytes present in most brain areas. PEA-15, a marker of cellular function or of cellular lineage? The PEA-15 expression profile in adult brain differs strikingly from that observed in vitro. In contrast to the in vivo situation, all cells express PEA-15 in primary astrocytes cultures. This could be the result of suppression of the regulations provided by the normal in situ environment of astrocytes. This hypothesis is supported by the data obtained with the G93A SOD1 mutant that demonstrate that PEA-15 does not behave as a product of a “housekeeping gene” but as a protein modulated according to environmental changes. This modulation of PEA-15 expression in the SOD1 mutant mouse may be related to its functions. In this mouse, as well as in other ALS models, motor neurons degeneration is accompanied with the up-regulation of numerous cytokines and trophic factors capable to activate members of the tumor necrosis factor receptor 1 (TNFR1) family of death receptors that are expressed by astrocytes, and/or to trigger the entrance into the cell cycle (Boillee et al., 2003; Hensley et al., 2003). PEA-15 is endowed with antiapoptotic and anti-proliferation properties in astrocytes and other non-CNS cell types, at least in vitro (Condorelli et al., 1999; Kitsberg et al., 1999; Formstecher et al., 2001; Xiao et al., 2002). It interacts with DED containing molecules such as FADD and caspase-8, this interaction taking place in the death inducing signaling complex that is located at the cell membrane. Such an

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interaction correlates with the inhibition of cell death induced by the activation of members of the TNFR1 family (Kitsberg et al., 1999; Xiao et al., 2002). Thus, PEA-15 expression may be related to cells exposed to death-inducing proteins in pathological as well as in physiological situations. Indeed, members of the TNFR1 family do not exclusively mediate apoptosis. They can also stimulate cell proliferation, cytokine production, and even neuritogenesis through ERK activation (OwenSchaub et al., 1993; Freiberg et al., 1997; Tsutsui et al., 1999; Shinohara et al., 2000; Desbarats et al., 2003). At present, it is not clear how the engagement of members of the TNFR1 family bypasses an apoptotic signal and promotes the other signals. It is nevertheless known that ERK activation by TNF family members has a dominantnegative effect on the apoptotic signaling by all these receptors (Tran et al., 2001). Besides its capacity to inhibit apoptosis through its interaction with DED-containing proteins, PEA-15 interacts also with ERK. PEA-15 binds to the kinase, exports it out of the cell nucleus, redirecting thus ERK activity toward its cytoplasmic rather than its nuclear substrates with, as a result, the limitation of cell proliferation (Formstecher et al., 2001). PEA-15 could thus be at the core of the mechanisms that influence the pro-apoptotic versus the other effects of the TNFR1 family of receptors. Altogether, these data support the hypothesis that PEA-15 expression is dependent on the functional status of the cell and on the stimuli to which it is exposed. The differential astrocyte expression of PEA-15 in embryonic cultures and in the adult brain raises however the alternative possibility that PEA-15 expression is related to the differentiation status of the cell. Expression of the protein in cells localized in areas of neurogenesis supports such a view. In the adult, PEA-15 expression was detected in the RMS and the SVZ. The adult SVZ contains progenitor cells, which proliferate, migrate along the RMS and differentiate in interneurons as they reach the olfactory bulbs (Doetsch et al., 1999). In the RMS, no co-localization of PEA-15 and GFAP was observed in the cells presenting the previously described typical “chain arrangement” of the migrating progenitors. These cells are thus likely to correspond to progenitors migrating toward the olfactory bulb. In the SVZ, the tightly packed organization of the cells precluded the detection of cells potentially labeled with both GFAP and PEA-15. In addition, we observed a dense PEA-15immunolabelling of neurogenesis zones in the murine embryonic brain (A. Sharif, unpublished observations). PEA-15 expression could in this context be switched on in cells that stop proliferating and enter the differentiation process. In addition, the presence of PEA-15 in areas of neurogenesis of the adult brain (Tramontin et al., 2003; Gritti, 2002) raises the possibility that, as suggested for cells expressing the LIM domain containing protein LMO1 (Scotti Campos, 2003), the subsets of astrocytes and neurons expressing PEA-15 derive from similar pools of progenitors.

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CONCLUSION This study demonstrates that astrocytes as well as neurons are cellular sources of PEA-15 in the adult brain, with the pattern of PEA-15 expression defining novel subpopulations of astrocytes throughout the brain. The widespread expression of the protein in subsets of astrocytes in most brain areas, its increased expression in pathological situations, associated with the known properties of PEA15, suggest that PEA-15 expression identifies cells challenged with potentially apoptotic and/or proliferative signals. Further analysis of PEA-15 expression under pathological conditions may help to elucidate its function in the adult brain. Acknowledgements—We are grateful to Dr. Serge Marty and Prof. Josette Cadusseau for their help in analysis of the results, Patrick Tierney for proofreading the manuscript and Prof. Jacques Glowinski for his constant support. We thank Amelia Dias-Morais for her expert technical help in genotyping. This research was supported by the Association pour la Recherche contre le Cancer (ARC, grant to H.C.) and ECOS program (L.B. and H.C.).

REFERENCES Araujo H, Danziger N, Cordier J, Glowinski J, Chneiweiss H (1993) Characterization of PEA-15, a major substrate for protein kinase C in astrocytes. J Biol Chem 268:5911–5920. Barres BA, Chun LL, Corey DP (1990) Ion channels in vertebrate glia. Annu Rev Neurosci 13:441–474. Bignami A, Dahl D (1974) Astrocyte-specific protein and neuroglial differentiation: an immunofluorescence study with antibodies to the glial fibrillary acidic protein. J Comp Neurol 153:27–38. Boillee S, Peschanski M, Junier MP (2003) The wobbler mouse: a neurodegeneration jigsaw puzzle. Mol Neurobiol 28:65–106. Brunet A, Roux D, Lenormand P, Dowd S, Keyse S, Pouyssegur J (1999) Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 18:664 –674. Chneiweiss H, Glowinski J, Premont J (1985) Modulation by monoamines of somatostatin-sensitive adenylate cyclase on neuronal and glial cells from the mouse brain in primary cultures. J Neurochem 44:1825–1831. Chung YH, Joo KM, Shin CM, Lee YJ, Shin DH, Lee KH, Cha CI (2003) Immunohistochemical study on the distribution of insulin-like growth factor I (IGF-I) receptor in the central nervous system of SOD1(G93A) mutant transgenic mice. Brain Res 994:253–259. Condorelli G, Vigliotta G, Cafieri A, Trencia A, Andalo P, Oriente F, Miele C, Caruso M, Formisano P, Beguinot F (1999) PED/PEA-15: an anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis. Oncogene 18:4409 –4415. Condorelli G, Vigliotta G, Iavarone C, Caruso M, Tocchetti CG, Andreozzi F, Cafieri A, Tecce MF, Formisano P, Beguinot L, Beguinot F (1998) PED/PEA-15 gene controls glucose transport and is overexpressed in type 2 diabetes mellitus. EMBO J 17:3858 –3866. Dahl D, Bignami A (1974) Heterogeneity of the glial fibrillary acidic protein in gliosed human brains. J Neurol Sci 23:551–563. Danziger N, Yokoyama M, Jay T, Cordier J, Glowinski J, Chneiweiss H (1995) Cellular expression, developmental regulation, and phylogenic conservation of PEA-15, the astrocytic major phosphoprotein and protein kinase C substrate. J Neurochem 64:1016 –1025. Denis-Donini S, Glowinski J, Prochiantz A (1984) Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurones. Nature 307:641–643. Desbarats J, Birge RB, Mimouni-Rongy M, Weinstein DE, Palerme JS,

Newell MK (2003) Fas engagement induces neurite growth through ERK activation and p35 upregulation. Nat Cell Biol 5:118 –125. Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703–716. Eddleston M, Mucke L (1993) Molecular profile of reactive astrocytesimplications for their role in neurologic disease. Neuroscience 54: 15–36. Eng LF (1988) Regulation of glial intermediate filaments in astrogliosis. In: The biochemical pathology of astrocytes (Norenberg MD, et al, eds), pp 79 –90. New York: Alan R. Liss, Inc. Estelles A, Charlton CA, Blau HM (1999) The phosphoprotein protein PEA-15 inhibits Fas- but increases TNF-R1-mediated caspase-8 activity and apoptosis. Dev Biol 216:16 –28. Estelles A, Yokoyama M, Nothias F, Vincent JD, Glowinski J, Vernier P, Chneiweiss H (1996) The major astrocytic phosphoprotein PEA-15 is encoded by two mRNAs conserved on their full length in mouse and human. J Biol Chem 271:14800 –14806. Feeney SJ, Austin L, Bennett TM, Kurek JB, Jean-Francois MJ, Muldoon C, Byrne E (2003) The effect of leukaemia inhibitory factor on SOD1 G93A murine amyotrophic lateral sclerosis. Cytokine 23: 108 –118. Formstecher E, Ramos JW, Fauquet M, Calderwood DA, Hsieh JC, Canton B, Nguyen XT, Barnier JV, Camonis J, Ginsberg MH, Chneiweiss H (2001) PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase. Dev Cell 1:239 –250. Freiberg RA, Spencer DM, Choate KA, Duh HJ, Schreiber SL, Crabtree GR, Khavari PA (1997) Fas signal transduction triggers either proliferation or apoptosis in human fibroblasts. J Invest Dermatol 108:215–219. Graeber MB, Streit WJ, Kreutzberg GW (1988) Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells. J Neurosci Res 21:18 –24. Gritti A, Bonfanti L, Doetsch F, Caille I, Alvarez-Buylia A, Lim DA, Galli R, Verdugo JM, Herrera DG, Vescovi AL (2002) Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents. J Neurosci 22:437–445. Gurney ME (1994) Transgenic-mouse model of amyotrophic lateral sclerosis. N Engl J Med 331:1721–1722. Hensley K, Fedynyshyn J, Ferrell S, Floyd RA, Gordon B, Grammas P, Hamdheydari L, Mhatre M, Mou S, Pye QN, Stewart C, West M, West S, Williamson KS (2003) Message and protein-level elevation of tumor necrosis factor alpha (TNF alpha) and TNF alphamodulating cytokines in spinal cords of the G93A-SOD1 mouse model for amyotrophic lateral sclerosis. Neurobiol Dis 14:74 –80. Hirano A (1996) Neuropathology of ALS: an overview. Neurology 47:S63–66. Howland DS, Liu J, She Y, Goad B, Maragakis NJ, Kim B, Erickson J, Kulik J, DeVito L, Psaltis G, DeGennaro LJ, Cleveland DW, Rothstein JD (2002) Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS). Proc Natl Acad Sci USA 99:1604 –1609. Israel JM, Schipke CG, Ohlemeyer C, Theodosis DT, Kettenmann H (2003) GABAa receptor-expressing astrocytes in the supraoptic nucleus lack glutamate uptake and receptor currents. Glia 44:102– 110. Kitsberg D, Formstecher E, Fauquet M, Kubes M, Cordier J, Canton B, Pan G, Rolli M, Glowinski J, Chneiweiss H (1999) Knock-out of the neural death effector domain protein PEA-15 demonstrates that its expression protects astrocytes from TNFalpha-induced apoptosis. J Neurosci 19:8244 –8251. Kubes M, Cordier J, Glowinski J, Girault JA, Chneiweiss H (1998) Endothelin induces a calcium-dependent phosphorylation of PEA-15 in intact astrocytes: identification of Ser104 and Ser116 phosphorylated, respectively, by protein kinase C and calcium/ calmodulin kinase II in vitro. J Neurochem 71:1307–1314. Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC (1995) Differential expression of two glial glutamate transporters in

A. Sharif et al. / Neuroscience 126 (2004) 263–275 the rat brain: quantitative and immunocytochemical observations. J Neurosci 15:1835–1853. Levine JB, Kong J, Nadler M, Xu Z (1999) Astrocytes interact intimately with degenerating motor neurons in mouse amyotrophic lateral sclerosis (ALS). Glia 28:215–224. Ma YJ, Berg-von der Emde K, Moholt-Siebert M, Hill DF, Ojeda SR (1994) Region-specific regulation of transforming growth factor alpha (TGF alpha) gene expression in astrocytes of the neuroendocrine brain. J Neurosci 14:5644 –5651. Matthias K, Kirchhoff F, Seifert G, Huttmann K, Matyash M, Kettenmann H, Steinhauser C (2003) Segregated expression of AMPAtype glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus. J Neurosci 23:1750 –1758. McCarthy KD, Salm AK (1991) Pharmacologically-distinct subsets of astroglia can be identified by their calcium response to neuroligands. Neuroscience 41:325–333. Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201–211. Muller T, Kettenmann H (1995) Physiology of Bergmann glial cells. Int Rev Neurobiol 38:341–359. Newbery HJ, Abbott CM (2001) Neuropathology of ALS: an overview. Trends Genet 17:S2–6. Nolte C, Matyash M, Pivneva T, Schipke CG, Ohlemeyer C, Hanisch UK, Kirchhoff F, Kettenmann H (2001) GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue. Glia 33:72–86. Norenberg MD (1994) Astrocyte responses to CNS injury. J Neuropathol Exp Neurol 53:213–220. Owen-Schaub LB, Meterissian S, Ford RJ (1993) Fas/APO-1 expression and function on malignant cells of hematologic and nonhematologic origin. J Immunother 14:234 –241. Poopalasundaram S, Knott C, Shamotienko OG, Foran PG, Dolly JO, Ghiani CA, Gallo V, Wilkin GP (2000) Glial heterogeneity in expression of the inwardly rectifying K(⫹) channel, Kir4.1, in adult rat CNS. Glia 30:362–372. Porter JT, McCarthy KD (1996) Hippocampal astrocytes in situ respond to glutamate released from synaptic terminals. J Neurosci 16:5073–5081. Rabchevsky AG, Weinitz JM, Coulpier M, Fages C, Tinel M, Junier MP (1998) A role for transforming growth factor alpha as an inducer of astrogliosis. J Neurosci 18:10541–10552. Ramos JW, Hughes PE, Renshaw MW, Schwartz MA, Formstecher E, Chneiweiss H, Ginsberg MH (2000) Death effector domain protein PEA-15 potentiates Ras activation of extracellular signal receptoractivated kinase by an adhesion-independent mechanism. Mol Biol Cell 11:2863–2872. Reuss B, Dono R, Unsicker K (2003) Functions of fibroblast growth factor (FGF)-2 and FGF-5 in astroglial differentiation and bloodbrain barrier permeability: evidence from mouse mutants. J Neurosci 23:6404 –6412. Ridet JL, Malhotra SK, Privat A, Gage FH (1997) Reactive astrocytes:

275

cellular and molecular cues to biological function. Trends Neurosci 20:570 –577. Scotti Campos L (2003) Evidence for astrocyte heterogeneity: a distinct subpopulation of protoplasmic-like glial cells is detected in transgenic mice expressing Lmo1-lacZ. Glia 43:195–207. Shinohara H, Yagita H, Ikawa Y, Oyaizu N (2000) Fas drives cell cycle progression in glioma cells via extracellular signal-regulated kinase activation. Cancer Res 60:1766 –1772. Stichel CC, Muller CM, Zilles K (1991) Distribution of glial fibrillary acidic protein and vimentin immunoreactivity during rat visual cortex development. J Neurocytol 20:97–108. Tibbetts MD, Zheng L, Lenardo MJ (2003) The death effector domain protein family: regulators of cellular homeostasis. Nat Immunol 4:404 –409. Tramontin AD, Garcia-Verdugo JM, Lim DA, Alvarez-Buylla A (2003) Postnatal development of radial glia and the ventricular zone (VZ): a continuum of the neural stem cell compartment. Cereb Cortex 13:580 –587. Tran SE, Holmstrom TH, Ahonen M, Kahari VM, Eriksson JE (2001) MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors. J Biol Chem 276:16484 –16490. Trencia A, Perfetti A, Cassese A, Vigliotta G, Miele C, Oriente F, Santopietro S, Giacco F, Condorelli G, Formisano P, Beguinot F (2003) Protein kinase B/Akt binds and phosphorylates PED/PEA15, stabilizing its antiapoptotic action. Mol Cell Biol 23:4511–4521. Tsutsui H, Kayagaki N, Kuida K, Nakano H, Hayashi N, Takeda K, Matsui K, Kashiwamura S, Hada T, Akira S, Yagita H, Okamura H, Nakanishi K (1999) Caspase-1-independent, Fas/Fas ligandmediated IL-18 secretion from macrophages causes acute liver injury in mice. Immunity 11:359 –367. Vaidyanathan H, Ramos JW (2003) RSK2 activity is regulated by its interaction with PEA-15. J Biol Chem 278:32367–32372. Walz W (2000) Controversy surrounding the existence of discrete functional classes of astrocytes in adult gray matter. Glia 31:95– 103. Walz W, Lang MK (1998) Immunocytochemical evidence for a distinct GFAP-negative subpopulation of astrocytes in the adult rat hippocampus. Neurosci Lett 257:127–130. Wilkin GP, Marriott DR, Cholewinski AJ (1990) Astrocyte heterogeneity. Trends Neurosci 13:43–46. Wong PC, Rothstein JD, Price DL (1998) The genetic and molecular mechanisms of motor neuron disease. Curr Opin Neurobiol 8:791– 799. Xiao C, Yang BF, Asadi N, Beguinot F, Hao C (2002) Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells. J Biol Chem 277:25020 –25025. Zhang Y, Redina O, Altshuller YM, Yamazaki M, Ramos J, Chneiweiss H, Kanaho Y, Frohman MA (2000) Regulation of expression of phospholipase D1 and D2 by PEA-15, a novel protein that interacts with them. J Biol Chem 275:35224 –35232.

(Accepted 13 February 2004) (Available online 6 May 2004)