Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.

Semaphorin proteins, although initially characterized as repulsive neuronal guidance cues, are now appreciated as major contributors to morphogenesis and homeostasis for a wide range of tissue types. Semaphorin-mediated long- and short-range repulsive, and attractive, guidance has profound influences on cellular morphology. The diversity of semaphorin receptor complexes utilized by various semaphorin ligands, the ability of semaphorins themselves to serve as receptors, and the myriad of intracellular signaling components that comprise semaphorin signaling cascades all contribute to cell-type-specific responses to semaphorins. Analysis of the molecular and cellular mechanisms underlying semaphorin function in neural and vascular systems provides insight into principles governing how this large protein family contributes to organogenesis, function, and disease.

The majority of excitatory synapses in the mammalian CNS are formed on dendritic spines1, and spine morphology and distribution are critical for synaptic transmission2–6, synaptic integration and plasticity7. Here, we show that a secreted semaphorin, Sema3F, is a negative regulator of spine development and synaptic structure. Mice with null mutations in genes encoding Sema3F, and its holoreceptor components neuropilin-2 (Npn-2) and plexinA3 (PlexA3), exhibit increased dentate gyrus (DG) granule cell (GC) and cortical layer V pyramidal neuron spine number and size, and also aberrant spine distribution. Moreover, Sema3F promotes loss of spines and excitatory synapses in dissociated neurons in vitro, and in Npn-2−/− brain slices cortical layer V and DG GCs exhibit increased mEPSC frequency. In contrast, a distinct Sema3A–Npn-1/PlexA4 signaling cascade controls basal dendritic arborization in layer V cortical neurons but does not influence spine morphogenesis or distribution. These disparate effects of secreted semaphorins are reflected in the restricted dendritic localization of Npn-2 to apical dendrites and of Npn-1 to all dendrites of cortical pyramidal neurons. Therefore, Sema3F signaling controls spine distribution along select dendritic processes, and distinct secreted semaphorin signaling events orchestrate CNS connectivity through the differential control of spine morphogenesis, synapse formation, and the elaboration of dendritic morphology.

Numerous cytoplasmic adaptor proteins, including JIP1, FE65, and X11␣, affect amyloid precursor protein (APP) processing and A␤ production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP ␤-C-terminal fragment and secreted A␤. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the ␤-C-terminal fragment of APP and A␤. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands. Proteolysis of the amyloid precursor protein (APP)2 results in production of the A␤ peptide, found in plaques of Alzheimer disease. The cytoplasmic domain of APP contains an NPTY sequence that serves as a binding motif for adaptor proteins that possess a phosphotyrosine binding domain (PTB), such as members of the Fe65, X11, JIP, and Dab protein families. Such adaptor proteins play critical roles in tyrosine kinase-mediated signal transduction, protein trafficking and localization, phagocytosis, cell fate determination, and neuronal development (1). Most importantly for Alzheimer disease, interactions between these cytoplasmic proteins and APP also lead to altered processing of APP.The effects of several of these adaptor proteins on APP trafficking and processing have been studied. The Fe65 family (FE65, FE65L1 (2), and FE65L2 (3)) is expressed at high levels in neurons (4). Co-expression of FE65 and APP promotes secreted APP and A␤ secretion in H4 cells and Madin-Darby canine kidney cells (2). However, co-expression of FE65 and APP in HEK293 cells stabilizes immature APP and inhibits APPs formation and A␤ secretion (5). The X11 family (X11␣, -␤, and -␥; also called Mint1, -2, and -3) are important in neuronal synaptic function (6). X11␣ slows cellular APP processing and reduces A␤40 and A␤42 secretion, perhaps through preventing trafficking of APP to subcellular compartments containing active ␥-secretase (7). X11␤ reduces A␤ levels and amyloid plaque formation in the brains of transgenic APP mice (8). Similar to X11␣ and -␤, JIP-1b interaction with APP stabilizes immature APP and inhibits APPs, A␤40, and A␤42 secretion in vitro (9).In contr...

Dopaminergic neurons in the mesodiencephalon (mdDA neurons) make precise synaptic connections with targets in the forebrain via the mesostriatal, mesolimbic, and mesoprefrontal pathways. Because of the functional importance of these remarkably complex ascending axon pathways and their implication in human disease, the mechanisms underlying the development of these connections are of considerable interest. Despite extensive in vitro studies, the molecular determinants that ensure the perfect formation of these pathways in vivo remain mostly unknown. Here, we determine the embryonic origin and ontogeny of the mouse mesoprefrontal pathway and use these data to reveal an unexpected requirement for semaphorin 3F (Sema3F) and its receptor neuropilin-2 (Npn-2) during mdDA pathway development using tissue culture approaches and analysis of sema3F Ϫ / Ϫ , npn-2 Ϫ / Ϫ , and npn-2 Ϫ / Ϫ ;TH-Cre mice. We show that Sema3F is a bifunctional guidance cue for mdDA axons, some of which have the remarkable ability to regulate their responsiveness to Sema3F as they develop. During early developmental stages, Sema3F chemorepulsion controls previously uncharacterized aspects of mdDA pathway development through both Npn-2-dependent (axon fasciculation and channeling) and Npn-2-independent (rostral growth) mechanisms. Later on, chemoattraction mediated by Sema3F and Npn-2 is required to orient mdDA axon projections in the cortical plate of the medial prefrontal cortex. This latter finding demonstrates that regulation of axon orientation in the target field occurs by chemoattractive mechanisms, and this is likely to also apply to other neural systems. In all, this study provides a framework for additional dissection of the molecular basis of mdDA pathway development and disease.

We report a cooperation between the neural adhesion molecule close homolog of L1 (CHL1) and the semaphorin 3A (Sema3A) receptor, neuropilin 1 (Npn1), important for establishment of area-specific thalamocortical projections. CHL1 deletion in mice selectively disrupted the projection of somatosensory thalamic axons from the ventrobasal (VB) nuclei, causing them to shift caudally and target the visual cortex. At the ventral telencephalon, an intermediate target with graded Sema3A expression, VB axons were caudally shifted in CHL1 Ϫ embryos and in Npn1 SemaϪ/Ϫ mutants, in which axons are nonresponsive to Sema3A. CHL1 colocalized with Npn1 on thalamic axons, and associated with Npn1 through a sequence in the CHL1 Ig1 domain that was required for Sema3A-induced growth cone collapse. These results identify a novel function for CHL1 in thalamic axon responsiveness to ventral telencephalic cues, and demonstrate a role for CHL1 and Npn1 in establishment of proper targeting of specific thalamocortical projections.

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.

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