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First published online 15 June 2005
doi: 10.1242/dev.01893

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SMAD pathway mediation of BDNF and TGFß2 regulation of proliferation and differentiation of hippocampal granule neurons

¹ NeuroAdaptations Group, Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, D-80804 Munich, Germany
² Neuroscience Group, Life and Health Sciences Research Institute (ICVS), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal

View larger version (61K): [in a new window]   Fig. 1. Cerebellar `stop' versus hippocampal `go' signals. (A) Characteristics of hippocampal and cerebellar neuronal cultures under basal conditions, showing expression of the neuronal markers TuJ1, MASH1 and MATH1, and levels of apoptosis (TUNEL, dark brown) and proliferation (BrdU incorporation, blue-black). (B,C) Exposure of hippocampal cells to CMCerebellum reduces BrdU incorporation (B); exposure of cerebellar cultures to CMHippocampus stimulates BrdU uptake (C). (D) In hippocampal-cerebellar slice co-cultures, hippocampal cell proliferation is reduced in the presence of cerebellar slices, whereas cerebellar cell proliferation is slightly increased in the presence of hippocampal tissue. Dose dependency of CMCerebellum anti-mitotic effects in hippocampal cultures are shown in E; the peptidergic nature of the anti-proliferative activity present in CMCerebellum is indicated by the fact that anti-proliferative activity is lost after boiling CMCerebellum. CMCerebellum also exerts anti-proliferative effects on hippocampus-derived HiB5 cells (F). Scale bar: 50 µm. Numerical data refer to mean±s.d. (n=4-6) *P<0.05, **P<0.01, ***P<0.001 (versus appropriate controls).

View larger version (22K): [in a new window]   Fig. 2. Hippocampal cultures express markers of cell cycle inhibition and neuronal differentiation after incubation in CMCerebellum. (A-D) Western blots and/or immunofluorescence staining show that addition of CMCerebellum (10 µl/ml) to hippocampal cultures induces expression of the cyclin-dependent kinase inhibitors p21 and p27 (A) and of the mature neuron marker MAP2A/MAP2B (B,C) with concomitant decreases in the expression of the neuroblast marker, doublecortin (D). Insets show examples of immunoblots for p21 (A) and p27 (A) and MAP2A/MAP2B (B), as well as for actin (which served as an internal reference). Results shown in A-D were obtained after 24 hours treatment with CMCerebellum. (E) CMCerebellum treatment influences proliferation and differentiation of neurons as shown by the significantly increased number of MAP2A/MAP2B-positive/BrdU-positive double-stained cells relative to the total number of BrdU-positive cell population. For this analysis, cells growing in CMCerebellum were exposed to BrdU for 8 hours, and washed and maintained in CMCerebellum for 72 hours before fixing and processing for MAP2A/MAP2B and BrdU double immunocytochemistry. There was no statistical difference on the incidence of apoptosis in cultures grown in either control medium or CMCerebellum. Numerical data refer to mean±s.d. (n=4-6) *P<0.05, **P<0.01, ***P<0.001 (versus appropriate controls).

View larger version (35K): [in a new window]   Fig. 3. Immunoneutralization of candidate anti-proliferative and differentiating factors in CMCerebellum and replication of effects by exogenous BDNF and TGFß2. (A) CMCerebellum was preadsorbed with the indicated dilutions of anti-NGF, BDNF or TGFß2. (B) Immunoneutralization of BDNF and TGFß2 significantly attenuates the anti-proliferative actions of CMCerebellum; anti-TGFß2 significantly attenuates the pro-differentiating effects of CMCerebellum, assessed by MAP2A/MAP2B expression. (C,D) Exogenous BDNF (C) and TGFß2 (D) dose-dependently inhibit BrdU retention in hippocampal cells; the inset (C), shows that transient expression of pBDNF also reduces BrdU incorporation (pEGFP used as transfection control). (E) Exogenous BDNF and TGFß2 promote neuronal maturation in hippocampal cultures (increased expression of MAP2A/MAP2B and neurons with neurite lengths more than twice the diameter of the soma). The control data in E, shown as 100%, represent 126/865 MAP2-positive cells (14.5%) with `long neurites' in the BDNF studies, and 94/518 MAP2-positive cells (18.2%) with `long neurites' in the TGFß2 experiments. Numerical data refer to mean±s.d. (n=4-6) *P<0.05, **P<0.01, ***P<0.001 (versus appropriate controls).

View larger version (34K): [in a new window]   Fig. 4. Involvement of TRKB/TGFß-RII and TGFß2 signaling pathways in the anti-mitotic and differentiating effects of CMCerebellum. (A) Transfection of cells with a dominant-negative form () of TRKB (TRKB) or TGFß receptor II (TGFß-RII) before treatment with CMCerebellum resulted in a significant increase in proliferation (versus controls transfected with GFP) (100% refers to BrdU incorporation in absence of CMCerebellum). (B) Results from cells treated as described above show that inhibition of expression of TGFß-RII or TRKB prevents CMCerebellum-induced neuronal differentiation, as measured by number of MAP2A/MAP2B-positive cells (cells not exposed to CMCerebellum provide the reference value of 100%). (C) Exposure of hippocampal cells to CMCerebellum, as well as to either TGFß2 (1 ng/ml) or BDNF (100 ng/ml) induces nuclear translocation of the TGFß2-specific partner SMAD2 and of co-SMAD4 within 3 hours, as shown by transient transfection experiments. (D,E) Introduction of dominant-negative forms of either SMAD3 (which specifically couples with TGFß2) or of Co-SMAD4 abrogates the anti-mitotic (D) and differentiating (E) effects of CMCerebellum on primary hippocampal cells; these changes do not reflect apoptosis as the number of TUNEL-stained and activated caspase 3 cells were not altered by the treatment (data not shown). (F-H) CMCerebellum stimulates generation of luciferase from the TGFß reporter gene 3TP-Lux in HiB5 cells (F), an effect abrogated when expression of either SMAD3 or Co-SMAD4 is blocked by transfection with their respective dominant-negative () forms (G); similarly, transactivation of 3TP-Lux is blocked in the presence of TGFß-RII and TRKB (H). Each half set of data in A,B,D,E were obtained in independent experiments in which GFP was used to control for between-culture variability in transfection efficiency. Data in G,H represent ratios of luciferase expression (fold change in treatments versus non-treated cells). In all cases, transfections were performed 24 hours before addition of CMCerebellum, TGFß2 or BDNF for 24 hours, after which the analysis was performed. Numerical data refer to mean±s.d. (n=4-6) *P<0.05, **P<0.01, ***P<0.001 (versus appropriate controls).

View larger version (25K): [in a new window]   Fig. 5. Mechanisms underlying the anti-mitotic and differentiating effects of CMCerebellum and BDNF – possible opportunities for crosstalk with TGFß signaling pathways. The anti-proliferative effects of BDNF (measured by BrdU incorporation) are significantly attenuated in primary hippocampal cells expressing a dominant-negative form of TGFß-RII. (B,C) The MEK inhibitor PD98059 (0.1 µM) counteracts the anti-proliferative (B) and pro-differentiating (C) effects of BDNF. (D) Transient transfection of HiB5 cells with pBDNF results in increased TGFß2 reporter gene (3TP-Lux) expression, an effect that is significantly attenuated by co-transfecting dominant-negative forms () of either TRKB, TGFß-RII or SMAD4, or by pretreatment with PD98059. (E) BDNF dose-dependently increases TGFß2 protein content in primary hippocampal cells; numerical data refer to mean±s.d. (n=4-6) *P<0.05, **P<0.01, ***P<0.001 (versus appropriate controls).