Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca 2؉ channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca 2؉ channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca 2؉ current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. influx coincides with synapse formation, we sought to identify proteins that might modulate Ca 2ϩ channel behavior and transmitter release during synaptogenesis. A proteomic screen of growth cone-associated proteins (see supplemental Fig. S1) and proteins interacting with the ␣1B subunit of CaV2.2 (8) identified collapsin response mediator protein-2 (CRMP-2) as a candidate CaV2.2-interacting protein.CRMPs are intracellular phosphoproteins implicated in neuronal growth cone advance and migration (9 -13). In neurons, CRMPs are expressed in the lamellipodia and filopodia of growth cones, in the shafts of axons, and in cell bodies. They contribute to axon formation by binding to tubulin heterodimers and promoting assembly of microtubules (14). CRMP phosphorylation causes dissociation from microtubules, leading to axonal outgrowth arrest (10,15,16). Overexpression of CRMP-2 in cultured hippocampal neurons induces extra axons (12), whereas CRMP-3/4/5 have been shown to be necessary for neurite extension (10), branching (17), and growth cone formation (18). The breadth of interactions of CRMP proteins with motor proteins, kinases, enzymes, and endocytosis-exocytosis-related proteins suggests that CRMPs may serve as adaptors/scaffold molecules (10). Although neurotransmission was not affected in CRMP-1 knock-out mice (19), long term potentiation, spatial learning, and memory were affected in both CRMP-1 and CRMP-3 knock-out mice (19,20), suggesting a role for CRMPs in neurotransmission as well as synaptogenesis. As potential binding partners, CRMPs could affect neurotransmission by a physical interaction with either the synaptic machinery or CaV2.2 itself.

Recent studies demonstrated that stromal cells isolated from adult bone marrow have the competence of differentiating into neuronal cells in vitro and in vivo. However, the capacity of marrow stromal cells or mesenchymal stem cells (MSCs) to differentiate into diverse neuronal cell populations and the identity of molecular factors that confer marrow stromal cells with the competence of a neuronal subtype have yet to be elucidated. Here, we show that Sonic hedgehog (Shh) and retinoic acid (RA), signaling molecules secreted from tissues in the vicinity of peripheral sensory ganglia during embryogenesis, exert synergistic effects on neural-competent MSCs to express a comprehensive set of glutamatergic sensory neuron markers. Application of Shh or RA alone had little or no effect on the expression of these neuronal subtype markers. In addition, incubation of MSCs with embryonic hindbrain͞somite͞ otocyst conditioned medium or prenatal cochlea explants promoted up-regulation of additional sensory neuron markers and process outgrowth. These results identify Shh and RA as sensory competence factors for adult pluripotent cells and establish the importance of interactions between adult pluripotent cells and the host microenvironment in neuronal subtype specification.

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VEGF receptor 1 (VEGFR-1͞Flt-1) is a high-affinity tyrosine kinase (TK) receptor for VEGF and regulates angiogenesis as well as monocyte͞macrophage functions. We previously showed that the osteoclast deficiency in osteopetrotic Csf1 op ͞Csf1 op (op͞op) mice is gradually restored in an endogenous, VEGF-dependent manner. However, the molecular basis of the recovery is still not clear. To examine which VEGFR is important and to clarify how colonystimulating factor 1 (CSF-1) and VEGF signals interact in osteoclastogenesis, we introduced a VEGFR-1 signaling deficiency (Flt1 TK؊/؊ ) into op͞op mice. The original Flt1 TK؊/؊ mice showed mild osteoclast reduction without bone marrow suppression. The double mutant (op͞opFlt1 TK؊/؊ ) mice, however, exhibited very severe osteoclast deficiency and did not have numbers of osteoclasts sufficient to form the bone marrow cavity. The narrow bone marrow cavity in the op͞opFlt1 TK؊/؊ mice was gradually replaced with fibrous tissue, resulting in severe marrow hypoplasia and extramedullary hematopoiesis. In addition to osteoclasts, osteoblasts also decreased in number in the op͞opFlt1 TK؊/؊ mice. These results strongly suggest that the interaction of signals by means of VEGFR-1 and the CSF-1 receptor plays a predominant role not only in osteoclastogenesis but also in the maintenance of bone marrow functions.osteoblast ͉ hematopoiesis ͉ hematopoietic niche ͉ osteopetrosis ͉ op͞op mouse

Wnt/β-catenin signaling promotes neural differentiation by activation of the neuron-specific transcription factors, Neurogenin1 (Ngn1), NeuroD and Brn3a, in the nervous system. Since neurons in cranial sensory ganglia and dorsal root ganglia transiently express Ngn1, NeuroD and Brn3a during embryonic development, we hypothesized that Wnt proteins could instructively promote a sensory neuronal fate from mesencymal stem cells (MSCs) directed to differentiate into neurons. Consistent with our hypothesis, Wnt1 induced expression of sensory neuron markers including Ngn1, NeuroD and Brn3a, as well as glutamatergic markers in neurally-induced MSCs in vitro and promoted engraftment of transplanted MSCs in the inner ear bearing selective loss of sensory neurons in vivo. Given the consensus function of T cell leukemia 3 (Tlx3), as a glutamatergic selector gene, we postulated that the effects of canonical Wnt signaling on sensory neuron and glutamatergic marker gene expression in MSCs may be mediated by Tlx3. We first confirmed that Wnt1 indeed up-regulates Tlx3 expression, which can be suppressed by canonical Wnt inhibitors. Next, our chromatin immunoprecipitation assays revealed that T-cell factor 3/4 (TCF3/4), Wnt-activated DNA binding proteins, interact with a regulatory region of Tlx3 in MSCs after neural induction. Furthermore, we demonstrated that forced expression of Tlx3 in MSCs induced sensory and glutamatergic neuron markers after neural induction. Together, these results identify Tlx3 as a novel target for canonical Wnt signaling that confers somatic stem cells with a sensory neuron phenotype upon neural induction.

A novel bone-resorbing factor was cloned using an expression cloning technique, which involved a Xenopus oocyte expression system and an assay for osteoclast formation. A candidate clone was isolated from a BW5147 mouse T-lymphoma cell cDNA library. Sequencing analysis identified the factor as ␥-glutamyltranspeptidase (GGT), which is an enzyme involved in glutathione metabolism. The addition of purified GGT protein to mouse bone marrow culture effectively induced formation of osteoclasts. An antibody against GGT inhibited osteoclast formation but not the enzymatic activity. We also demonstrated that an inactive form of GGT, the enzymatic activity of which had been blocked by chemical modification with a specific inhibitor, acivicin, supported osteoclast formation. These results indicate that GGT acts on osteoclast formation independent of its own enzymatic activity. Furthermore, both native GGT and inactive GGT stimulated the expression of the receptor activator of nuclear factor-B ligand (RANKL) mRNA and protein from bone marrow stromal cells. This report is the first demonstration of a novel biological activity of GGT protein in a manner independent of its enzymatic activity.

These findings may have important clinical implications as a means of delivering MSCs in the cochlea for stem-cell replacement therapy. Survival of transplanted MSCs into the modiolus of the cochlea may result in regeneration of damaged SGNs.

Retinoic acid signaling plays essential roles in morphogenesis and neural development through transcriptional regulation of downstream target genes. It is believed that the balance between the activities of synthesizing and metabolizing enzymes determines the amount of active retinoic acid to which a developing tissue is exposed. In this study, we investigated spatiotemporal expression patterns of four synthesizing enzymes, the retinaldehyde dehydrogenases 1, 2, 3, and 4 (Raldh1, Raldh2, Raldh3, and Raldh4) and two metabolizing enzymes (Cyp26A1 and Cyp26B1) in the embryonic and postnatal mouse inner ear by using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization, and Western blot analysis. Quantitative RT-PCR analysis and Western blot data revealed that the expression of CYP26s was much higher than that of Raldhs at early embryonic ages but that Cyp26 expression was downregulated during embryonic development. Conversely, the expression levels of Raldh2 and -3 increased during development and were significantly higher than the Cyp26 levels at postnatal day 20. At this age, Raldh3 was expressed predominantly in the cochlea, whereas Raldh2 was present in the vestibular end organ. At early embryonic stages, as observed by in situ hybridization, the synthesizing enzymes were expressed only in the dorsoventral epithelium of the otocyst, whereas the metabolizing enzymes were present mainly in mesenchymal cells surrounding the otic epithelium. At later stages, Raldh2, Raldh3, and Cyp26B1 were confined to the stria vascularis, spiral ganglion, and supporting cells in the cochlear and vestibular epithelia, respectively. The downregulation of Cyp26s and the upregulation of Raldhs after birth during inner ear maturation suggest tissue changes in the sensitivity to retinoic acid concentrations.