Pro- and mature brain-derived neurotrophic factor (BDNF) activate two distinct receptors: p75 neurotrophin receptor (p75(NTR)) and TrkB. Mature BDNF facilitates hippocampal synaptic potentiation through TrkB. Here we report that proBDNF, by activating p75(NTR), facilitates hippocampal long-term depression (LTD). Electron microscopy showed that p75(NTR) localized in dendritic spines, in addition to afferent terminals, of CA1 neurons. Deletion of p75(NTR) in mice selectively impaired the NMDA receptor-dependent LTD, without affecting other forms of synaptic plasticity. p75(NTR-/-) mice also showed a decrease in the expression of NR2B, an NMDA receptor subunit uniquely involved in LTD. Activation of p75(NTR) by proBDNF enhanced NR2B-dependent LTD and NR2B-mediated synaptic currents. These results show a crucial role for proBDNF-p75(NTR) signaling in LTD and its potential mechanism, and together with the finding that mature BDNF promotes synaptic potentiation, suggest a bidirectional regulation of synaptic plasticity by proBDNF and mature BDNF.

Alternatively spliced brain-derived neurotrophic factor (BDNF) transcripts are targeted to distinct cellular compartments in neurons but the mechanisms underlying this sorting are unknown. Although only some BDNF isoforms are targeted to dendrites, we have found that the coding region common to all BDNF transcripts contains a constitutively active dendritic targeting signal and that this signal is suppressed in transcripts containing exons 1 or 4, which are restricted to the cell soma and proximal dendrites. This dendritic targeting signal is mediated by translin, an RNA-binding protein implicated in RNA trafficking, and is disrupted by the G196A mutation associated with memory deficits and psychiatric disorders. Molecular modeling and mutational studies indicate that the G196A mutation blocks dendritic targeting of BDNF mRNA by disrupting its interaction with translin. These findings implicate abnormal dendritic trafficking of BDNF mRNA in the pathophysiology of neuropsychiatric disorders linked to the G196A mutation.neuropsychiatric disorders ͉ neurotrophins S everal lines of evidence indicate that targeting of BDNF mRNA to dendrites plays a key role in mediating synaptic plasticity (1-4). However, the molecular mechanisms regulating this process and the differential subcellular localization of alternatively spliced BDNF transcripts, remain to be clarified.Multiple BDNF transcripts are generated by alternative splicing of one 5Ј exon with a shared 3Ј exon containing the entire BDNF coding region and either a short or long 3Ј UTR sequence (5, 6). In recent studies, we have demonstrated that BDNF transcripts differ in their subcellular localization (7). Exon 1 and 4 transcripts are localized in the cell soma, while exon 2 and 6 transcripts show a somato-dendritic localization. Thus, splice variants appear to encode spatial localization signals used to preferentially regulate BDNF expression in different subcellular domains (2, 3). A recent study has suggested that the long 3Ј UTR contains signals necessary for dendritic targeting of BDNF transcripts (4). However, it is unlikely that this mechanism can fully account for the differential dendritic targeting displayed by BDNF transcripts because more than one-third of exon 4 transcripts, which are retained in the soma, contain the long 3Ј UTR. Conversely, more than one-half of exon 6 transcripts, an isoform that displays targeting to dendrites, contain the short 3Ј UTR. To help define the mechanisms underlying differential localization of BDNF transcripts, we have tested the hypothesis that additional signals might be encoded by other BDNF mRNA regions.

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The cellular distribution of GABAC receptor rho1 and rho2 subunits in the rat central nervous system remains controversial. We investigated how these subunits were distributed in cerebellum, hippocampus and spinal cord at postnatal day 1, 7 or in adult life. We found that in the adult cerebellum rho1 and rho2 mRNAs were expressed in Purkinje cells and basket-like cells only. In the hippocampus both subunits were expressed throughout the CA1 pyramidal layer, dentate gyrus and scattered interneurons with maximum staining intensity at P7. In the adult hippocampus in situ staining was predominantly found on interneurons. GABAC antibody labelling in P7 and adult hippocampus was largely overlapping with the in situ staining. Western blot analysis showed GABAC receptor in retina, ovary and testis. In the spinal cord the rho2 signal was consistently stronger than rho1 with overlapping expression patterns. At P1, the most intensely labelled cells were the motoneurons while on P7 and adult sections, interneurons and motoneurons were likewise labelled. On spinal neurons both rho1 and rho2 mRNAs showed somatodendritic localization, extending out for >100 microm with punctate appearance especially in adult cells. A similar spinal distribution pattern was provided with polyclonal antibody labelling, suggesting close correspondence between mRNA and protein compartmentalization. Electrophysiological experiments indicated that P1 spinal motoneurons did possess functional GABAC receptors even though GABAC receptors played little role in evoked synaptic transmission. Our results suggest a pattern of rho1 and rho2 subunit distribution more widespread than hitherto suspected with strong developmental regulation of subunit occurrence.

Gas6 is a growth factor related to protein S that was identified as the ligand for the Axl receptor tyrosine kinase (RTK) family. In this study, we show that Gas6 induces a growth response in a cultured mammalian mammary cell line, C57MG. The presence of Gas6 in the medium induces growth after confluence and similarly causes cell cycle reentry of density-inhibited C57MG cells. We show that Axl RTK but not Rse is efficiently activated by Gas6 in density-inhibited C57MG cells. We have analyzed the signaling required for the Gas6 proliferative effect and found a requirement for PI3K-, S6K-, and Ras-activated pathways. We also demonstrate that Gas6 activates Akt and concomitantly inhibits GSK3 activity in a wortmannin-dependent manner. Interestingly, Gas6 induces up-regulation of cytosolic ␤-catenin, while membrane-associated ␤-catenin remains unaffected. Stabilization of ␤-catenin in C57MG cells is correlated with activation of a T-cell factor (TCF)-responsive transcriptional element. We thus provide evidence that Gas6 is mitogenic and induces ␤-catenin proto-oncogene stabilization and subsequent TCF/Lef transcriptional activation in a mammary system. These results suggest that Gas6-Axl interaction, through stabilization of ␤-catenin, may have a role in mammary development and/or be involved in the progression of mammary tumors.

IntroductionOsteoprotegerin (OPG) is a soluble member of the tumor necrosis factor (TNF) receptor superfamily, whose best characterized activity is the inhibition of receptor activator of NF-B ligand (RANKL)-stimulated formation of osteoclasts. 1 OPG also interacts with TNF-related apoptosis-inducing ligand (TRAIL), 2 a deathinducing ligand whose extracellular domain shares a 35% homology with RANKL. Mounting evidence indicates that the ability of OPG to inhibit TRAIL cytotoxicity might represent an important mechanism in promoting the survival of prostate cancer, breast cancer, colon cancer, and multiple myeloma cells at least in vitro. [3][4][5] More importantly, for the purpose of this study, it has also been shown that OPG is produced in vitro by vascular endothelial cells, is overexpressed by tumor-associated endothelial cells, and is able to promote the survival/proliferation of endothelial cells in a paracrine or autocrine manner. [6][7][8][9][10] Moreover, elevated levels of serum OPG have been detected in both solid tumors and hematologic malignancies. 11 Although the relative contribution of both normal and tumor-associated endothelial cells to serum OPG remains to be established, 2 both the stromal and the tumoral vasculature is engaged in extensive interactions with the cancer cells, probably contributing to the growth and invasiveness of the tumor. 12,13 The p53 protein is a sequence-specific transcription factor that functions as a major tumor suppressor in mammals. 13,14 In response to various types of oncogenic stresses, p53 is activated to promote cell-cycle exit, apoptosis, or replicative senescence, thereby preventing the propagation of incipient cancer cells.Consequently, p53 is very often disabled within cancer cells, either by direct mutational inactivation of the TP53 gene or by alterations in other genes of which the products impinge on p53. Whereas the effect of p53 has been widely investigated in a cell-autonomous context, much less attention has been given to its non-cell-autonomous functions, although it has been recently demonstrated that the stromal compartment of the tumors is able to modulate the latency of tumorigenesis in a p53-dependent manner. 12,13 On these bases, the aim of this study was to investigate the effect of p53 knock-down and/or induction on the expression and release of OPG in human endothelial cells. Methods Cell cultures and treatmentsHuman umbilical vein endothelial cells (HUVECs) were purchased from BioWhittaker (Walkersville, MD) and grown on 0.2% gelatin-coated tissue culture plates in M199 endothelial growth medium supplemented with 20% fetal bovine serum, 10 g/mL heparin, and 50 g/mL ECGF (endothelial cell growth factor; all from BioWhittaker). In all experiments, cells were used between the 3rd and 5th passages in vitro, as previously described. 15 For endothelial cell treatments, the following reagents have been used: Nutlin-3 (Cayman Chemical, Ann Arbor, MI), TNF-␣ (R&D Systems, Minneapolis, MN), and Aphidicolin (Alexis Biochemicals, Lausen, Switzerland...

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising natural anticancer therapeutic agent because through its death receptors, TRAIL-R1 and TRAIL-R2, it induces apoptosis in many transformed tumor cells, but not in the majority of normal cells. Hence, agonistic compounds directed against TRAIL death receptors have the potential of being excellent cancer therapeutic agents, with minimal cytotoxicity in normal tissues. Here, we report the selection and characterization of a new single-chain fragment variable (scFv) to TRAIL-R2 receptor isolated from a human phage-display library, produced as minibody (MB), and characterized for the in vitro anti-leukemic tumoricidal activity. The anti-TRAIL-R2 MB2.23 efficiently and specifically bound to membrane-associated TRAIL-R2 on different leukemic cell lines and could act as a direct agonist in vitro, initiating apoptotic signaling as well as complement-dependent cytotoxicity and antibody-dependent cell cytotoxicity, providing a rationale for further investigations of MB2.23 in anticancer therapy.