Shc signaling in differentiating neural progenitor cells - Nature

© 2001 Nature Publishing Group http://neurosci.nature.com

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Shc signaling in differentiating neural progenitor cells Luciano Conti1, Simonetta Sipione1, Lorenzo Magrassi2,3, Luca Bonfanti4, Dorotea Rigamonti1, Valentina Pettirossi5, Marc Peschanski6, Bassam Haddad6, PierGiuseppe Pelicci5, Gabriele Milanesi3,7, Giuliana Pelicci5 and Elena Cattaneo1 1 Department of Pharmacological Sciences, Center of Excellence on Neurodegenerative Diseases, University of Milano, Via Balzaretti 9, 20133 Milano, Italy 2 Department of Neurosurgery, Department of Surgery, University of Pavia–I.R.C.C.S. Policlinico S. Matteo, P. le Golgi 2, 27100 Pavia, Italy 3 Istituto di Genetica, Biologia ed Evoluzionistica, CNR, via Abbiategrasso 207, 27100 Pavia, Italy 4 Department of Morfofisiologia Veterinaria, University of Torino, via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy 5 European Institute of Oncology, via Ripamonti 435, 20141 Milano, Italy 6 Faculté de Médecine, INSERM U421, 8 rue du Général Sarrail, 94010 Créteil, France 7 Department of Biology and Genetics for Medical Sciences, University of Milano, via Viotti 5, 20133 Milano, Italy

Correspondence should be addressed to E.C. ([email protected])

Previously we found that the availability of ShcA adapter is maximal in neural stem cells but that it is absent in mature neurons. Here we report that ShcC, unlike ShcA, is not present in neural stem/progenitor cells, but is expressed after cessation of their division and becomes selectively enriched in mature neurons. Analyses of its activity in differentiating neural stem/progenitor cells revealed that ShcC positively affects their viability and neuronal maturation via recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway. We suggest that the switch from ShcA to ShcC modifies the responsiveness of neural stem/progenitor cells to extracellular stimuli, generating proliferation (with ShcA) or survival/differentiation (with ShcC).

The ventricular zone central nervous system (CNS) stem/progenitor cells in the developing brain undergo extensive cell division to generate diverse cell types1,2. Previous work demonstrated that these events are regulated by diffusible factors, most of which recruit the MAPK pathway and act via protein tyrosine kinase receptors (RPTK)2,3. The Shc adapter proteins are important triggers of these pathways because, once phosphorylated, they function as connectors between the activated RPTK and downstream signaling molecules3,4. The Shc gene, recently renamed ShcA, encodes three proteins of 46 (p46ShcA), 52 (p52 ShcA) and 66 (p66ShcA) kDa, each containing an SH2-phosphotyrosine binding domain, a central glycine and proline-rich region, and a second phosphotyrosine binding site named the PTB domain5,6. ShcA is very broadly expressed outside the CNS and its importance is indicated by the early embryonic lethal phenotype of p52ShcA null mutation7 and by the increase in life span and resistance to stress stimuli of p46ShcA-null mutant animals8. Consistent with the prominent role in signal transduction, ShcA adapter proteins were suggested to be ubiquitously expressed 3. However, our analysis of CNS tissue at different developmental stages revealed that ShcA is limited to the germinal epithelium of the embryonic brain, where the immature neural stem/progenitor cells actively divide9. Concurrent with neurogenesis, ShcA proteins and mRNAs became strictly downregulated, both temporally and spatially9. When these data are considered in light of studies on nonneuronal cells, showing that ShcA is activated by mitogenic growth factors, the implication is that ShcA may be involved in the division of neural stem cells3,9. Consistently, mature neurons are almost devoid of ShcA9. This finding contrasts with a large nature neuroscience • volume 4 no 6 • june 2001

body of evidence showing that growth factors and the MAPK pathway are essential well after development. The discovery of a gene specifically enriched in adult brain tissue10–12 and homologous to ShcA, named ShcC, prompted further analysis of its presence and function during differentiation. We show here that, unlike ShcA, ShcC was not expressed in dividing neural stem/progenitor cells but was selectively found in postmitotic and mature neurons. ShcC critically affected survival and phenotypic maturation of these cells. Indeed, we found that ShcC overexpression in primary neural progenitors increased their survival and differentiation, and ablation of endogenous ShcC activity in mature neurons by transfected SH2 domain of ShcC (possibly via a dominant negative effect) induced cell death. We also report that these effects depended on recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway, because pharmacological and molecular inhibition of these two pathways abolishes ShcC-driven survival and differentiation, respectively. We conclude that the switch from ShcA to ShcC is critical for proper neuronal development, allowing neural stem/progenitor cells to proliferate in the presence of ShcA and to survive/differentiate in the presence of ShcC.

RESULTS ShcC replaces ShcA in maturing neural progenitor cells Previous studies showed that in coincidence with neurogenesis, ShcA levels are sharply downregulated, both temporally and spatially, with the adult brain devoid of ShcA9. The lack of expression of ShcA in mature neurons suggested that other proteins are 579



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Fig. 1. ShcC replaces ShcA in maturing a b CNS neurons. (a) Time course of ShcC expression during brain maturation. The western blot shows that the two immunoreactive bands (52 and 66 kDa) corresponding to ShcC isoforms (bottom) are expressed with an inverse pattern with respect to ShcA isoforms (top). Immunostaining with anti α-Tubulin is shown. E, embryonic; P, postnatal. One of three experiments that generated the same result is shown. (b, c) ShcC is not c d expressed in proliferating neural progenitor cells. FACS profile of E15 brain cells stained with antibodies against ShcA (FITC channel) and BrdU (Phycoerythrin channel) (b) or ShcC (Phycoerythrin channel) and BrdU (FITC channel) (c). Approximately 47% of the cells were double labeled by BrdU and ShcA (b, right, UR quadrant). On the contrary, all BrdU-positive cells were ShcC negative (c, right, LR quadrant). In (b) and (c), left graphs represent controls where the primary antibodies were omitted (LL quadrant). UL, upper left; UR, upper right; LL, lower left; LR, lower right. (d) ShcC is expressed in mature neurons in the adult rodent brain. Cerebral cortex (top left and middle), cerebellar cortex (top right, bottom left), hippocampus (bottom, middle) and primary olfactory cortex (bottom right). The labeling is restricted to the cytoplasm and initial dendrites of mature neurons, with a punctate distribution (see high magnification). ShcC immunoreactivity appears to be particularly abundant in large-sized projection neurons, such as pyramidal neurons and Purkinje neurons, but is also detectable in the granule cell layers of the cerebellum (g) and hippocampus, which contain small-sized interneurons. A widespread punctate distribution appears in the granule cell layer (g), whereas in the molecular layer (m), the labeling is restricted to some neuronal cell bodies of basket cells and to dendrites of Purkinje cells. a, field CA4 of Ammon’s horn; g, granule cell layer of the cerebellar cortex and of the dentate gyrus; p, Purkinje cell layer. Scale bars, 20 µm.

involved in signaling from activated growth factor receptors in these cells3. The two recently discovered homologues, ShcB and ShcC, were obvious candidates; ShcC was particularly obvious, as its presence is restricted to the mature brain10–12. We thus analyzed ShcC expression at different developmental brain stages and found that ShcC protein levels increase progressively during brain maturation, with an expression pattern opposite to that of ShcA (Fig. 1a). The two immunoreactive bands of 52 and 66 kDa, which correspond to the two rodent ShcC isoforms, were found at very low levels in early developmental stages, but were maximally expressed in the postnatal and mature brain (Fig. 1a, bottom; top, identical membrane reacted with an anti-ShcA antibody, expected profile)9. To analyze whether ShcC becomes expressed only in postmitotic neural cells, we performed a two-color FACS analysis on cells from the rat developing CNS. These cells were isolated from the telencephalic vesicles of BrdU-exposed E15 rat embryos followed by double labeling with anti-BrdU and anti-ShcA or antiShcC antibodies. In agreement with the western blot data, BrdU-positive cells were labeled only by the ShcA antibody (Fig. 1b) but not by the ShcC antibody (Fig. 1c). This data suggests that ShcC becomes expressed only after cessation of proliferation of neural stem/progenitor cells. To further confirm these data, and to identify the cells expressing ShcC in vivo, we performed an immunohistochemical study at different developmental stages. Consistent with the western blot and FACS data (Fig. 1a–c), sections from animals at embryonic stages did not reveal any staining, whereas only post-mitotic cells were labeled at postnatal stages (data not shown). Particularly, immunohistochemical analysis at adult stages revealed that ShcC is restricted to mature neurons (Fig. 1d). Glial 580

cells exhibited no appreciable ShcC immunoreactivity. Furthermore, all neuronal cell types in different brain regions were immunoreactive, indicating that ShcC is not restricted to particular subpopulations of neurons. To confirm these observations in a system where neuronal differentiation could be precisely controlled, we derived neural progenitor cells from human fetal telencephalon and cultured them for one (undifferentiated condition) or seven days (differentiated condition) in serum-free media (Fig. 2a). Analysis of Shc content before and after differentiation revealed that changes in ShcA and ShcC levels similar to those observed in vivo occurred during the in vitro differentiation of the human neural progenitor cells (Fig. 2b). As already demonstrated in vivo in rodents9, levels of Grb2 adapter protein remained unchanged (Fig. 2b). Taken together, these data demonstrate that important changes in Shc content occur when immature neural stem/progenitor cells undergo neuronal differentiation. This alternative expression of the Shc could underlie two possible mechanisms: ShcA and ShcC are differently recruited by mitogenic/differentiation-inducing ligands, or ShcA and ShcC are similarly activated by these ligands but their specific expression at immature and mature stages differently instructs downstream signaling molecules. To try to discriminate between these two possibilities, we analyzed ShcA and ShcC activation in proliferating neural stem/progenitor cells and post-mitotic primary neurons following a 15-minute stimulation with ligands whose effects on neural progenitors and post-mitotic neurons are well documented3,4. We found that both ShcA and ShcC can be equally recruited by the same ligands (Fig. 2c), anticipating that regulation of their availability during brain development may represent the key event that drives proliferation or differentiation signals. nature neuroscience • volume 4 no 6 • june 2001



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Fig. 2. ShcA and ShcC expression a b c and activation in neural progenitor cells. (a) Immunocytochemical characterization of progenitor cell cultures from human fetal brain in vitro for one day (undifferentiated condition, left column) to obtain immature neural progenitor cells (as assessed by BrdU incorporation, second row) or for seven days (right column) to give rise to post-mitotic neurons (as confirmed by lack of BrdU staining, second row). The composition of the cultures before and after differentiation was confirmed by immunocytochemistry using anti-nestin (third row) and anti-βIII-tubulin (fourth row) antibodies. Arrows point to BrdU-positive cells. Scale bar, 10 µm. (b) Variations in ShcA and ShcC levels in cultured neural progenitor cells from human telencephalon. The western blot was done on lysates from human neural progenitor cells (one day in vitro) and differentiated human primary neurons derived from fetal telencephalon (seven days in vitro). Inverse expression patterns occur for ShcA (left) and ShcC (center), whereas Grb2 (right) levels remain invariant. One of four experiments that generated the same results is shown. (c) Growth factor stimulations lead to similar pattern of ShcA (top) and ShcC (bottom) tyrosine phosphorylation in cultures of neural progenitor cells or post-mitotic neurons. Membranes were immunoreacted with an anti-phosphotyrosine antibody. The amount of immunoprecipitated material was verified by reacting the filters with the appropriate antibody (lower autoradiograms in ShcA and ShcC blots).

ShcC increases survival and maturation The peculiar appearance of ShcC during development and its neuronally restricted localization prompted us to investigate the biological events in which ShcC might be involved, and the identity of the downstream signaling proteins it may activate. Therefore, we expressed the human p54 ShcC cDNA in neural progenitors or post-mitotic neurons obtained from the E14 rat striatum and grown in a minimal medium. The p54ShcC cDNA was cloned in the pIRES2-eGFP, an IRES (internal ribosome entry site)-containing vector for bicistronic expression of ShcC and eGFP (enhanced green fluorescent protein). Coexpression was

confirmed by immunocytochemical analyses in transfected cultures (data not shown). We assayed the occurrence of cell death by evaluating the percentage of apoptotic (as confirmed by nuclear fragmentation evaluated by Hoechst staining) eGFP-positive neural progenitor cells at different days after transfection (Fig. 3). Cells transfected with control-eGFP plasmid progressively underwent apoptosis (Fig. 3a, left). On the contrary, expression of exogenous ShcC greatly diminished the occurrence of apoptotic cell death in the cultures (percentage of apoptotic cells among the eGFP positive, seven days after transfection, controleGFP transfected cultures, 68 ± 3.1; ShcC-transfected cells,

Fig. 3. ShcC increases survival and differentiation of neural progenitors. Cultures of neural progenitor cells were transfected with ShcC, SH2-ShcC DN or control vectors (all coexpressing eGFP). (a) Left, ShcC overexpression improves cell viability. Neural progenitor cells transfected with ShcC (black diamonds), SH2-ShcC DN (black circles) or eGFP control (black squares) vectors and visualized by the fluorescence of the eGFP. Cell viability was evaluated by counting the number of alive and apoptotic eGFP-positive cells in the cultures at different days after transfection. Middle, ShcC overexpression improves neurite length. Measurements were taken seven days after transfection in ShcC (white bars) and control (black bars) vector-transfected cells. Transfected cells were classified based on the presence of short (25–50 µm), middle a (50–100 µm), long (100–200 µm) or very long (200–300 µm) neurites. Data are expressed as the percentage of total live cells (n = 300) falling into these four categories. Right, ShcC overexpression increases the number of processes per b cell. Neurite formation was measured in ShcC (white bars) and in control (black bars) plasmid-transfected cells, seven days after transfection. Only cells with processes longer than 25 µm were considered. Data are expressed as the percentage of total live cells (n = 400) bearing one, two or three processes per cell. (b) Pictures of eGFP-expressing cells from cultures transfected with control (left), ShcC (middle) or SH2-ShcC DN (right) vectors, seven days after transfection. Bottom, corresponding Hoechst staining. Appearance of ShcC-positive cells is typically neuronal, with wellbranched arborization and long processes. Scale bar, 20 µm. nature neuroscience • volume 4 no 6 • june 2001

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Fig. 4. Manipulation of ShcC levels and a function affects survival and neurite elongation of primary neurons. Transfection, counting of transfected cells and measurement of neurite length and processes formation were done as described in Fig. 3. (a) Left, ShcC overexpression (black diamonds) improves b neuronal viability, whereas SH2-ShcC DN (black circles) induces apoptosis of primary post-mitotic neurons with respect to control (black squares) transfected cultures. Middle, ShcC overexpression (white bars) increases neurite length in post-mitotic neurons, as measured twelve days after transfection, with respect to control cultures (black bars). Right, ShcC overexpression (white bars) increases process formation (with respect to control cultures, black bars) as measured twelve days after transfection. (b) Pictures of eGFPexpressing neurons from cultures transfected with control (left), ShcC (middle) or SH2-ShcC DN (right) vectors twelve days after transfection. Bottom row, corresponding Hoechst pictures. ShcC-transfected neurons are highly ramified relative to control cells. An example of an apoptotic neuron transfected with the SH2-ShcC DN plasmid is shown. Condensed nucleus (thick arrow) and cell body fragmentation (thin arrow) are clearly visible. Scale bar, 20 µm.

36 ± 3.4; p < 0.001 by t-test). To further confirm the pro-survival effect of ShcC, we overexpressed an SH2-ShcC dominant negative (DN; black circles; SH2 portion of the protein, possibly evoking a dominant-negative effect13) in the same cultures and found a percentage of apoptotic cells slightly higher than that observed in control cultures (Fig. 3a, left). We conclude that ShcC signaling mediates the survival of maturing neural progenitor cells. In addition, our data indicate that the SH2 domain of the protein mediates this effect. In fact, transfection of a full-length ShcC lacking the SH2 domain in the same cultures failed to reduce cell death (percentage of apoptotic cells among the eGFP positive, seven days after transfection, 72 ± 4.1; p < 0.001 by t-test). We found that ShcC-transfected cells not only showed more survival but also showed enhanced neuronal features such as increased neurite elongation and the appearance of additional main processes, leading to a bipolar-like morphology (Fig. 3b). On the contrary, cells from control eGFP-transfected cultures displayed more immature neuronal morphology, as expected (Fig. 3b). Quantification of these parameters seven days after transfection showed a significant increase in neurites’ length and number in ShcC-transfected cells (Fig. 3a, middle and right, white bars) with respect to control (black bars). In particular, most ShcC-transfected cells had a neurite length in the range of 50–300 µm (with respect to 25–100 µm in the control cultures; Fig. 3a, middle) and showed two major processes (with respect to a single main process present in control cells, right graph; p < 0,001 by t-test; this analysis included only those control cells that survived and were most highly differentiated at this time point). A relevant portion of the ShcC-transfected cells had three main processes and a neurite length between 200–300 µm (Fig. 3a, middle and right). We conclude that besides promoting cell survival, ShcC also signals morphological differentiation of maturing neural progenitor cells. To further confirm these functions of ShcC, cells were allowed to differentiate into post-mitotic neurons and were then transfected with control (pIRES2-eGFP), ShcC or SH2-ShcC DN vec582

tors or with a full-length ShcC construct deleted of the SH2 domain. Cell death was evaluated as described above. (The typical appearance of SH2-ShcC DN transfected neurons is reported in Fig. 4b.) As shown, nucleus (thick arrow) and cell body (thin arrow) are visibly fragmented, as also confirmed by Hoechst staining (Fig. 4b). Indeed, transfection of SH2-ShcC DN (black circles) in post-mitotic neurons resulted in a higher proportion of transfected neurons undergoing apoptosis (Fig. 4a, left; percentage of apoptotic cells among the eGFP positive, twelve days after transfection, control-eGFP-transfected cells, 61.2 ± 1.7; SH2-ShcC DN-transfected cells, 83.7 ± 3.4; p < 0.001 by t-test). In contrast, exogenous ShcC (black diamonds) reduced cell death at all time points analyzed. Finally, transfection of the full-length ShcC mutant lacking the SH2 domain was not able to reproduce the effect of the full-length molecule (percentage of apoptotic cells among the eGFP positive at three, eight and twelve days after transfection, 42 ± 2.8, 57 ± 3.3, 69 ± 5.2, respectively; p < 0.001 by t-test). As previously shown for the immature neural progenitor cells, ShcC-overexpressing neurons also enhanced their morphological differentiation (Fig. 4a, middle and right, white bars; Fig. 4b). These data indicate a prominent role for ShcC in postmitotic neurons. As confirmation of the specificity of the ShcC function, ShcA transfection in the same cultures failed to reproduce the effects of ShcC (data not shown). To investigate the molecular and biochemical mechanisms underlying ShcC’s role in differentiating neural progenitor cells, we retrovirally transfected conditionally immortalized ST14A cells14 with the human p54ShcC cDNA. Subclones of control (eGFPexpressing, ST14A-Co) and ShcC-positive (eGFP-ShcC-expressing, ST14A-ShcC) cells were isolated, and the presence of the exogenous protein was confirmed by western blot analysis (Fig. 5a) and immunocytochemistry (not shown). Whereas exposure of ST14A-Co cells (black squares, Fig. 5b) to 39°C, which is not permissive for cell growth (simulating a post-mitotic stage), resulted in progressive cell death15, ST14A-ShcC cells (black circles, nature neuroscience • volume 4 no 6 • june 2001



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Fig. 5. ShcC enhances viability and neuronal differena c b tiation of conditionally immortalized neural progenitor cells. (a) Retroviral transduction of ST14A cells generated control- (ST14A-Co) or ShcC- (ST14AShcC) expressing subclones as shown by western blot. Adult rat brain was used as positive control. (b) MTT assay on ST14A-Co (black squares) and ST14A-ShcC (black circles) cells. Values indicate the percentage of mitochondrial activity at the non-permissive temperature. Each point represents the mean ± s.d. of six experiments done on three different ST14A-Co clones and four ST14A-ShcC clones. Increased survival of ST14A-ShcC cells is shown. (c) Western blots showing NF-68 and PSD-95 expression in ST14A-Co and ST14A-ShcC cells. Increased levels of these antigens are found in ST14A-ShcC cells in post-mitotic condition. NG, normal growth; PC, post-mitotic conditions. Equal loading was confirmed by exposing the membranes to an anti α-tubulin antibody. One of four experiments that generated the same result is shown.

Fig. 5b) remained viable even after prolonged exposure to 39°C, and in the absence of cell division as confirmed by the lack of BrdU uptake (data not shown) 14,15. In the same conditions, ST14A-ShcC cells were also markedly immunopositive for antigens typically enriched in mature neurons (NF-68, MAP2, βIII-tubulin; Table 1). These results were confirmed by western blot analysis performed with anti-NF-68 and anti-PSD-95 antibodies (Fig. 5c), the latter being an antigen enriched in post-mitotic neurons16.

that survived revealed no apparent effect of the Akt inhibition on the differentiation elicited by ShcC (data not shown). Exposure of ST14A-ShcC cells to a pharmacological inhibitor of the Erks, PD 098059 (Fig. 6a), had no effect on cell viability, but it did influence their ability to induce differentation. In fact, in these conditions, the percentage of ST14A-ShcC cells immunoreactive to NF-68, MAP2 and βIII-tubulin returned to control levels (Table 1). The same results were obtained when expression of NF-68 and PSD-95 antigens was evaluated by western blot analysis (data not shown). We conclude that ShcC-driven MAPK activation positively ShcC modulates Akt, Bad and Erks phosphorylation influences aspects of neuronal differentiation. This was further In various non-neuronal systems, stimulation of RPTKs by mitodemonstrated by transfection of a MEK dominant-negative mutant gens leads to ShcA phosphorylation and subsequent activation (MEK KA97, specifically inhibiting Erks activation) or transfection of Ras and Erk-MAPKs (extracellular signal-regulated kinaseof the SH2-ShcC DN cDNA into ST14A-ShcC cells. Expression of mitogen activated protein kinases, namely p42Erk2 and p44Erk1)17. these cDNAs resulted in a 60% and 75% reduction, respectively, of Other downstream effectors of activated RPTKs lie along the the intensity of the PSD 95 immunoreactive bands with respect to PI3K (phosphatidylinositol 3-kinase) pathway, the activity of untreated ST14A-ShcC cells (Fig. 6b). which has a direct effect on cell survival18–20 via activation of the To investigate the molecular mechanisms by which ShcC influserine/threonine kinase Akt21,22. We therefore analyzed whether ences MAPK and PI3K activation, we analyzed the phosphorylathe observed biological effects driven by ShcC occur via recruittion state of the Erks and of the protein kinase Akt. We found that ment of the MAPK and/or of PI3K pathways. Akt was activated in ST14A-Co cells, but a stronger immunoreTo investigate the involvement of the PI3-K pathway in ShcCactive band corresponding to phosphorylated Akt was observed in promoted cell survival, ST14A-ShcC cells were exposed to the ST14A-ShcC cells (Fig. 6c). Even at longer time intervals pharmacological agents wortmannin and LY294002, two struc(24 hours), phosphorylated Akt was still detectable (Fig. 6c). Furturally unrelated PI3-K inhibitors (Fig. 6a). In both conditions, thermore, in ST14A-ShcC cells, Bad (a pro-apoptotic member of ST14A-ShcC cells exhibited a marked decrease in their survival. the Bcl2 family that is a major substrate for Akt and, when phosIn addition, blockade of Akt function by transfection of the Akt phorylated, is sequestered by 14-3-3 proteins resulting in cell surDN (K179M) mutant (or of the SH2-ShcC DN) significantly vival22–24) was found highly phosphorylated both in Ser112 and reduced cell survival (Fig. 6a). Co-transfection of this same construct with ShcC in post-mitotic primary neurons similarly abolSer 136 (Fig. 6c), thus suggesting that ShcC-driven pro-survival ished ShcC-driven cell survival (percentage of apoptotic cells effect results from inhibition of Bad pro-apoptotic activity. among the eGFP positive, twelve days after transfection, ShcCOn the other hand, p42Erk2 and p44Erk1 were almost equally transfected cultures, 45.7 ± 2.5; ShcC/Akt DN-cotransfected cells, susceptible to phosphorylation in ST14A-Co and ST14A-ShcC cells, 64.8 ± 4.1; p < 0.001 by t-test). Morphological analysis of the cells but exhibited a different pattern of activation (Fig. 6c). Thus, Erks were transiently phosphorylated in ST14ACo cells (with a peak at 30 min) but Table 1. ShcC increases the number of cells immunoreactive for neuronally remained phosphorylated for longer times enriched antigens. (up to 24 hours) in ST14A-ShcC cells. NF-68 MAP2 βIII-tubulin These results indicate that ShcC initiates Co (%) ShcC (%) Co (%) ShcC (%) Co (%) ShcC (%) signaling cascades leading to neuronal dif4 days at 39°C 4.1 ± 3 30.7 ± 2 5.1 ± 2.6 20.1 ± 4 7.1 ± 4 21.7 ± 5 ferentiation that requires prolonged stimulation of the MAPK. 7 days at 39°C 3.7 ± 2.5 33.3 ± 4 3.1 ± 3 16.7 ± 5 5.1 ± 2.8 15.9 ± 4 The role of ShcC-driven Akt and + PD 098059 0.9 ± 0.4 1.7 ± 1.2 1.1 ± 0.8 3.7 ± 2.3 1.4 ± 0.9 4.1 ± 3.5 MAPK activation is further demonstrated The number of ST14A-Co and ST14A-ShcC cells immunoreactive for neuronal-specific antigens was evalu- by the finding that transfection of the SH2ated by immunocytochemistry in cultures grown at 39°C (post-mitotic conditions) for four and seven days. At this latter time point, the few ST14A-Co cells that survived the temperature shift did not express ShcC DN construct, shown to abolish βIII-tubulin, MAP2 or NF-68 antigens. No GFAP immunoreactivity was observed in ST14A-Co or ST14AShcC-evoked biological effects, partially ShcC cells. Cells were exposed to PD 098059 as described in Fig. 6a. Data were confirmed in an indepenreduced Akt and Erks phosphorylation in dent experiment. Values are given as mean ± s.d. ST14A-ShcC cells (Fig. 6d).

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Fig. 6. ShcC modulates Akt and c a p42Erk2/p44Erk1 activation and Bad phosphorylation. (a) Inhibition of the PI3K signaling reduces ShcC-mediated cell viability. Cultures of ST14A-ShcC cells were grown for 3 days at 39°C and then exposed to PI3K (LY294002 and wortmannin) or MAPK (PD 098059) inhibitors for 48 h. Parallel ST14A-ShcC cultures were transfected with Akt DN (K179M) mutant or with the SH2-ShcC DN cDNAs. Mean ± s.d. of four experiments. (b) Inhibition of MAPK activity abolishes neuronal differentiation induced by ShcC presence. Western blots show PSD-95 expression in ST14A-ShcC cells in the absence or presence of transfected MEK dominant-negative mutant (MEK KA97) or of SH2-ShcC DN b d cDNAs. Equal loading was confirmed by exposing the filter to an anti α-tubulin antibody. (c) Modification of Akt, Bad and MAPK activities in presence of ShcC. Membranes were reacted with phospho-Akt (Ser473), phospho-Bad Ser112 and Ser136 or phosphop44/42 MAPK (Thr202/Tyr204) antibodies. Equal loading was confirmed by immunostaining with anti-Akt, anti-Bad and anti-Erks antibodies (lower autoradiograms in each antibody blots). One of three experiments that generated the same result is shown. (d) Expression of SH2-ShcC DN abolishes ShcC-mediated modification in Akt and MAPK activities. Analysis was done as described in (c).

DISCUSSION A precise understanding of the processes controlling maturation of mitotically active neural stem/progenitor cells into post-mitotic neurons requires investigation of intracellular signaling mechanisms. Here we show that during the transition from proliferation to neuronal differentiation, specific changes occur in the availability of the different members of the Shc family of adapter molecules. Previous work has explored the influence of diffusible factors that, by acting via RPTK and recruiting the Ras-MAPK pathway, regulate the phenotypic potential of neural stem/progenitor cells and/or neuronal stability1,2,25. The Shc adapter proteins are important mediators of these signaling events3. The Shc family consists of three genes named ShcA, ShcB and ShcC, each encoding different protein isoforms3. Most of the molecular, biochemical and biological analyses of Shc function have so far been conducted on ShcA molecules, which are tyrosine phosphorylated by activated RPTK, and which recruit the Grb2/SOS complex, ultimately resulting in Ras-MAPK activation and mitogenesis4,17. Our analyses of ShcA in the developing embryonic brain demonstrated that it is selectively expressed in the mitotically active germinal zone, and that its mRNAs and proteins are progressively downregulated during the differentiation of neural stem/progenitor cells to neurons9. ShcA is replaced by ShcC, which is not expressed in neural progenitors. ShcC appeared in post-mitotic neurons and reached maximal levels in the adult brain, where it was found localized only in neurons. Particularly, analyses of ShcC presence in various regions of the adult brain revealed that this adapter is widely expressed in all neuronal cells, suggesting key role(s) of ShcC in these cells. Such role(s) must be highly conserved, because similar changes in ShcA and ShcC levels during neuronal maturation have been observed in several mammalian species (rat, mouse and human9; Figs. 1 and 2). Given the central role of ShcA in signal transduction, we postulated that appearance of ShcC during neu584

ronal maturation results in different ‘connector functions’ compared with ShcA and that through initiation of different signaling cascades, it modifies the capability of differentiating neural stem/progenitor cells to respond to extracellular factors. The demonstration of the existence of a strict link between Shc levels and cell responsiveness comes from a study7 showing that ShcA expression and activity are required in cells of the cardiovascular system to make them responsive to low concentrations of growth factors. Indeed, although a low concentration of growth factors is necessary to activate the MAPK pathway in mouse embryo fibroblasts (MEF), cells from ShcA null mutant mice require an higher concentration of growth factors to activate the same signaling cascade7. Similarly, the highly dynamic changes in Shc protein levels occurring during brain development may critically influence cellular responsiveness and neuronal differentiation of neural stem cells. ShcC is expressed in post-mitotic neurons, where it acts to promote differentiation and improve survival. We found that ShcC increases the expression of neuronal antigens via prolonged stimulation of MAPK. This behavior is reminiscent of that described in PC12 cells exposed to NGF, where persistent activation of MAPK is required for neuronal differentiation26,27. On the contrary, ShcC-driven prosurvival effect occurs via recruitment of the PI3K-Akt-Bad pathway, as its inhibition reverts this effect. Furthermore, we showed that Akt activation due to ShcC presence causes phosphorylation (with inhibition) of both Ser136 and Ser112 in Bad, a proapoptotic member of the Bcl2 family23,24,28. Bad Ser136 is a known target of Akt24, whereas the Ser112 site is phosphorylated by the Rsks (MAPK-activated kinases)29,30. Although we found that both sites are phosphorylated in ShcC cells, inhibition of Akt activity alone greatly affected ShcC-promoted cell survival. Our results suggest that ShcC-driven survival of differentiating neural progenitor cells mainly results from PI3K-Akt-Bad (Ser136) pathway activation, as also seen in PC12 cells31. nature neuroscience • volume 4 no 6 • june 2001



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The results here presented unveil a scenario within which physiological changes in the availability of ShcA and ShcC adaptors during brain development modify neural stem/progenitor cell responsiveness as a function of the developing environment. Furthermore, based on this evidence, strategies for interfering with neuronal cell demise in neurodegenerative diseases should include consideration of ShcC levels and activity. While this paper was submitted, the presence of neuronal dysfunction in the peripheral nervous system of double ShcB/C null mice was reported32.

METHODS

Vector constructions. We subcloned p54ShcC cDNA into the multiple cloning site of the amphotrophic retroviral PINCO vector33 or in the bicistronic pIRES2-eGFP vector (Clontech, Palo Alto, California). The cDNAs for the p46/p52/p66ShcA isoforms were cloned in the bicistronic pIRES2-eGFP vector (Clontech). The SH2-ShcC DN was prepared as described13 and subcloned into pIRES2-eGFP. ShcC deleted from the SH2 region was obtained by taking advantage of KpnI site located at the beginning of the SH2 region. The KpnI fragment was then subcloned into pIRES2-eGFP vector in frame with a new stop codon. In Akt DN (K179M) mutant (Upstate Biotechnology, Lake Placid, New York), lysine 179 is converted to methionine, resulting in a loss of affinity for ATP. MEK dominant-negative (MEK KA97 mutant)30 was provided by A. Bonni and M. Greenberg. Culture and transfection of neural cells. Human telencephalic primary cells were isolated from fetuses at 8–9 weeks after conception. Cells were plated onto poly-ornithine in supplemented N2 serum-free medium9 and processed for immunocytochemistry after one or seven days. Striatum primordia were isolated from E14 rat brains. To analyze the effect of ShcC on differentiating neural progenitors, cells were mechanically dissociated and plated in supplemented N2 serum-free medium onto poly-ornithine-coated dishes. Plating density was 105 cells/cm2. Transfection was done the following day in 24-well plates using Lipofectamine reagent (Gibco, Rockville, Maryland) 34. The day after transfection, N2-unsupplemented DMEM-F12 serum-free medium was added (minimal medium). These culture conditions do not favor survival and differentiation of the cells. To analyze the role of ShcC in differentiated post-mitotic neurons, cells were dissociated as described above, plated in B27-supplemented Neurobasal medium35 and allowed to differentiate for 7 days. Cells were transfected and cultured in the same medium. These culture conditions are particularly suitable for long-term culturing of neurons32. Transfected cells were visualized by co-expression of eGFP. Cell viability was evaluated by counting the number of alive and apoptotic eGFP-positive cells at different days after transfection. We counted 400–600 cells per well, and the values were averaged from quadruplicate samples in three independent experiments. We counted the number of processes and measured neurite length using a calibrated function of NIH Image software. Culture, infection and transfection of ST14A progenitor cells. ST14A cells were grown routinely at 33°C in the presence of Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum14. Shifting to the non-permissive temperature of 39°C leads to degradation of the large T-antigen with consequent blockade of cell proliferation. ShcA isoforms proved to be the only Shc molecules carried by ST14A cells, their levels being downregulated at the non-permissive temperature. Under the same conditions (39°C), a faint band corresponding to the two ShcC isoforms could be detected. ST14A cells were subjected to two infection cycles (two hours each) with filtered conditioned medium from a retroviral packaging cell line transducing the PINCO vector alone or carrying the p54ShcC cDNA35. For inhibition experiments, LY294002 and wortmannin were applied at 10 µM and 5 µM, respectively. PD098059 was added at 20 µM. The compounds were reapplied every 24 hours. Transfections of ST14AShcC cultures were done at 33°C using Lipofectamine reagent (Gibco)34. The day after transfection, cultures were shifted to 39°C for seven days. Cell counting, MTT assay and BrdU measurement. Cells were plated in triplicates into 6-well plates at a density of 2 × 105/well. After an 8-h incubation at 33°C, the cultures were shifted to 39°C. At the times indicated, nature neuroscience • volume 4 no 6 • june 2001

cells were trypsinized, resuspended in 20 ml of Isoton solution and counted using a Coulter Counter ZM machine (Coulter Electronics, Luton, UK). MTT and BrdU incorporation assays were done as described15. FACS analysis. E15 brains of rat embryos were exposed to BrdU by 2 i.p. injections in the mother (100 mg BrdU per kg body weight) 4 and 2 h before embryo collection. Telencephalic vesicles were dissected free of the meninges, mechanically dissociated into a single-cell suspension, and fixed in 1% paraformaldehyde-0.01% Tween 20 (ref. 36). All flow cytometrical analyses were done on a FACSVantage flow cytometer (BectonDickinson, San Jose, California) and data were analyzed using Cell Quest software (Becton-Dickinson). Cells were stained overnight with an antiShcC mouse monoclonal antibody (1:50 dilution, Transduction Laboratories, Lexington, Kentucky), washed and incubated for 1 h in a goat anti-mouse phycoeritrin-conjugated antibody (1:20 dilution, Sigma, St. Louis, Missouri) followed by further washings and incubation in a rabbit anti-goat phycoeritrin-conjugated antibody (1:20 dilution, Sigma). After washing, the cells were digested for 30 min with 50 U/ml of DNase-I in Ca 2+ and Mg 2+ PBS (Sigma). At the end, the cells were washed again and incubated for 1 h at room temperature with a mouse monoclonal antibody against BrdU conjugated to FITC (1:10 dilution, Roche, Indianapolis, Indiana). Considering the timing of BrdU administration, both proliferating neural/stem progenitor cells and early postmitotic neurons are present in the fraction of BrdU-positive cells. A parallel set of cells was also stained overnight with an anti-ShcA rabbit polyclonal antibody (1:50 dilution, Transduction Laboratories) washed and reacted with a sheep anti-rabbit FITC-conjugated antibody (1:30 dilution, Sigma) followed by DNase I digestion and exposure to mouse monoclonal anti-BrdU antibody (1:100 dilution, Sigma) secondarily revealed with a goat anti-mouse phycoeritrin-conjugated antibody (1:20 dilution, Sigma). All rinses were done in the presence of 0.1 BSA (0.1%, Sigma) and Tween-20 (0.2%, Sigma). None of the primary or secondary antibodies exhibited nonspecific staining when tested on the appropriate controls. Immunostaining. Embryos were fixed by immersion in 4% paraformaldehyde (PFA); postnatal and adult Wistar rats were perfused intracardially with 4% PFA and 1% picric acid in 0.1 M sodium phosphate buffer, and post-fixed overnight in the same fixative. Brains were frozen, cryostat sectioned (10 µm thick) and processed for immunohistochemistry as previously described37,38. The antiserum against ShcC was diluted 1:2000 in PBS containing 0.2% BSA, 0.2% poly-lysine hydrobromide and 0.3% Triton X-100. A commercially available anti-ShcC antibody (Transduction Laboratories) was also used after a 1:50 dilution in the same buffer. These two antibodies exhibited the same neuronalspecific immunoreactivity and were tested for their specificity by immunoprecipitation and western blot assays11. Cultured cells were fixed with 4% PFA, permeabilized and incubated with anti-ShcC (1:100 dilution; Transduction Laboratories), MAP2 (1:200 dilution; Roche), anti-βIII-Tubulin (1:200 dilution; Promega, Madison, Wisconsin), anti-NF-68 (1:200 dilution; Transduction Laboratories) and anti-GFAP (1:200 dilution; Sigma) antibodies. Fluoresceinconjugated and TRITC-conjugated secondary antibodies (1:100 dilution) were applied for 1 h. Percentage of cells expressing cell type-specific marker was evaluated by immunocytochemistry done on cultures grown at 39°C for four and seven days. BrdU detection was done as described15. Positive cells were scored manually. Immunoprecipitation and western blotting procedures. For immunoprecipitation studies, cultures were starved overnight and stimulated with BDNF (50 ng/ml), NGF (50 ng/ml), NT3 (50 ng/ml) or EGF (25 ng/ml) for 15 min. After this period, cells were rinsed with PBS and processed as described9. Proteic lysates from brain tissues were prepared as described9. Immunoprecipitation was done using polyclonal anti-ShcA (Transduction Laboratories) and anti-ShcC antibodies11. For filter immunostaining, anti-phosphotyrosine polyclonal antibody (New England Biolabs, Beverly, Massachusetts, applied at 1:2000 dilution), anti-ShcC monoclonal antibody (Transduction Laboratories, applied at 1:5000 dilution) and anti-ShcA monoclonal antibody (Transduction Laboratories, applied at 1:1000 dilution) were used. For neuronal differentiation analyses, antiPSD-95 monoclonal antibody (Transduction Laboratories) and 585



articles

anti-NF-68 (Transduction Laboratories) were applied at 1:1000 dilution. Monoclonal anti-α-tubulin antibody (Sigma) was applied at 1:5000 dilution. For the analysis of Erks, cells were grown for three days at 39°C in complete medium, starved overnight in serum-free medium, and then stimulated with serum for 0 min (untreated control), 10 min, 30 min, 60 min, 4 h or 24 h. Then, the cultures were rinsed in PBS and lysed in lysis buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromophenol blue) at 4°C. Equal amounts of proteins were loaded onto 10% SDS-PAGE. Monoclonal anti-phosphoErk, antiphosphoBad Ser 112 and anti-phosphoBad Ser 136 and anti-phosphoAkt from New England Biolabs were applied at 1:2000, 1:1000 and 1:1000 dilutions, respectively. After washing with TBS-T, membranes were exposed to HRP-conjugated secondary antibodies (1:2000, Kirkegard and Perry Laboratory). Immunoreactivities were detected using the enhanced chemiluminescence method (ECL Plus, Amersham, Italy) according to manufacturer’s instructions. Membranes were stripped and immunostained with polyclonal anti Erk1 (Santa Cruz Biotechnology, Santa Cruz, California), anti-Bad (New England Biolabs) and anti-Akt (New England Biolabs), applied at 1:4000, 1:1000 and 1:1000 dilutions, respectively.

14. 15. 16. 17. 18. 19. 20. 21. 22.

ACKNOWLEDGEMENTS This work was supported by Associazione Italiana Ricerca Cancro and Ministry of University and Technological Research (Italy #9905261712) to E.C., by Telethon (Italy, E1025) to L.C., and Telethon (Italy, A128) and C.N.R. (P.S. Basi Biologiche Malattie Neurodegenerative) to L.M. We thank R. DeBona and M. De Simone for their help with the cell cultures and immunoreactions, and F. Benvenuto for help with the FACS analysis. We also thank P. Delli Santi for her technical support and L. Chiappino for her photographic expertise.

23. 24. 25. 26. 27.

RECEIVED 4 JANUARY; ACCEPTED 12 APRIL 2001

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nature neuroscience • volume 4 no 6 • june 2001