TrkB signaling modulates spine density and morphology independent of dendrite structure in cultured neonatal Purkinje cells.

Neurotrophins cooperate with neural activity to modulate CNS neuronal survival and dendritic differentiation. In a previous study, we demonstrated that a critical balance of neurotrophin and neural activity is required for Purkinje cell survival in cocultures of purified granule and Purkinje cells (Morrison and Mason, 1998). Here we investigate whether TrkB signaling regulates dendrite and spine development of Purkinje cells. BDNF treatment of purified Purkinje cells cultured alone did not elicit formation of mature dendrites or spines. In cocultures of granule and Purkinje cells, however, continuous treatment with BDNF over a 2 week postnatal culture period increased the density of Purkinje cell dendritic spines relative to controls without causing a shift in the proportions of headed and filopodia-like spines. The increase in spine number was blocked by adding TrkB-IgG to the medium together with BDNF. Although BDNF alone did not consistently modify the morphology of dendritic spines, treatment with TrkB-IgG alone yielded spines with longer necks than those in control cultures. None of these treatments altered Purkinje cell dendritic complexity. These analyses reveal a role for TrkB signaling in modulating spine development, consistent with recently reported effects of neurotrophins on synaptic function. Moreover, spine development can be uncoupled from dendrite outgrowth in this reductionist system of purified presynaptic and postsynaptic neurons.

Granule neuron regulation of Purkinje cell development: striking a balance between neurotrophin and glutamate signaling.

Granule neurons, presynaptic afferents of Purkinje cells, are potent regulators of Purkinje cell development. Purified Purkinje cells survive and differentiate poorly, whereas coculture with granule neurons enhances their survival and dendritic development. Here we investigate the role of neurotrophins in granule-Purkinje cell interactions. BDNF or NT-4 improves, but NT-3 or CNTF reduces, survival of isolated Purkinje cells. When granule neurons are present, however, BDNF or NT-4 treatment leads to Purkinje cell loss. This decrease is overcome by anti-BDNF or TrkB-IgG-blocking reagents or by CNQX, a non-NMDA glutamate receptor antagonist. Furthermore, BDNF increases the spine density on the surviving Purkinje cells. These results suggest that Purkinje cell survival and differentiation are context-dependent and require a balance between neurotrophin- and activity-dependent signaling.

Axon-target interactions in the developing cerebellum.

After entering target regions, afferent growth cones grow among putative target cells, stop extending upon meeting target cells, and transit into a synaptic ending. During these events, signals are transmitted to and from target cells to stimulate programs of differentiation. Here we describe three approaches to unraveling mechanisms of these phases of synaptogenesis. First, dye-labeling in the intact cerebellum has revealed the orchestration of afferent ingrowth and contacts with target cells. Second, an in vitro model based on purified granule neurons has shed light on the role of target cells in the arrest of afferent extension. Third, coculture of purified granule neurons (parallel fiber afferents) with purified Purkinje cells has demonstrated facets of afferent regulation of target cell differentiation. These analyses have suggested molecular mechanisms that mediate maturation of afferents and their targets.

Eph family receptors and their ligands distribute in opposing gradients in the developing mouse retina.

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal-low nasal and high ventral-low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.

Granulocyte-macrophage colony-stimulating factor signals for increased glucose uptake in human melanoma cells.

While the primary targets for granulocyte-macrophage colony-stimulating factor (GM-CSF) are hematopoietic precursors and mature myeloid cells, GM-CSF receptors (GMR) are also found on normal tissues including placenta, endothelium, and oligodendrocytes as well as certain malignant cells. The function of GMR in these nonhematopoietic cells is unknown. We studied the function of GMR in human melanoma cell lines. Six of seven cell lines tested (clones 1-5 and 3.44 of SK-MEL-131, SK-MEL-188, SK-MEL-23, SK-MEL-22, and SK-MEL-22A) expressed mRNA encoding the membrane-bound and soluble isoforms of the alpha subunit of the GMR. Melanoma cell lines in early stages of differentiation expressed the largest quantities of alpha-subunit mRNA. Although five of these lines expressed trace levels of mRNA encoding the beta subunit of the GMR, Scatchard analysis of equilibrium binding data derived from three of the cell lines showed that they expressed only low-affinity GMR. Clones 3.44 and 1-5 of SK-MEL-131, and SK-MEL-188 cells expressed receptors with a dissociation constant (kd) for GM-CSF in the following ranges: 0.7 to 0.8, 1.2 to 1.8, and 0.4 to 0.8 nmol/L, respectively. GM-CSF stimulated glucose uptake in four of the melanoma cell lines expressing the alpha subunit, presumably through facilitative glucose transporters, as uptake was blocked by cytochalasin B but not cytochalasin E. Stimulation of glucose uptake was transient, with maximum stimulation occurring at approximately 30 minutes in the presence of 1 nmol/L GM-CSF. GM-CSF stimulated glucose uptake 1.4- to 2.0-fold but did not stimulate cell proliferation. These results suggest a metabolic role for the low-affinity GMR in melanoma cell lines and indicate that the alpha subunit of the GMR can signal for increased glucose uptake in nonhematopoietic tumor cells.

Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication.

Poliovirus initiates infection of primate cells by binding to the poliovirus receptor, Pvr. Mouse cells do not bind poliovirus but express a Pvr homolog, Mph, that does not function as a poliovirus receptor. Previous work has shown that the first immunoglobulin-like domain of the Pvr protein contains the virus binding site. To further identify sequences of Pvr important for its interaction with poliovirus, stable cell lines expressing mutated Pvr molecules were examined for their abilities to bind virus and support virus replication. Substitution of the amino-terminal domain of Mph with that of Pvr yields a molecule that can function as a poliovirus receptor. Cells expressing this chimeric receptor have normal binding affinity for poliovirus, yet the kinetics of virus replication are delayed. Results of virus alteration assays indicate that this chimeric receptor is defective in converting native virus to 135S altered particles. This defect is not observed with cells expressing receptor recombinants that include Pvr domains 1 and 2. Because altered particles are believed to be an intermediate in poliovirus entry, these findings suggest that Pvr domains 2 and 3 participate in early stages of infection. Additional mutants were made by substituting variant Mph residues for the corresponding residues in Pvr. The results were interpreted by using a model of Pvr predicted from the known structures of other immunoglobulin-like V-type domains. Analysis of stable cell lines expressing the mutant proteins revealed that virus binding is influenced by mutations in the predicted C'-C" loop, the C" beta-strand, the C"-D loop, and the D-E loop. Mutations in homologous regions of the immunoglobulin-like CD4 molecule alter its interaction with gp120 of human immunodeficiency virus type 1. Cells expressing Pvr mutations on the predicted C" edge do not develop cytopathic effect during poliovirus infection, suggesting that poliovirus-induced cytopathic effect may be induced by the virus-receptor interaction.

Hemorrhagic tumor necrosis during a pilot trial of tumor necrosis factor-alpha and anti-GD3 ganglioside monoclonal antibody in patients with metastatic melanoma.

Hemorrhagic tumor necrosis is an inflammatory event that leads to selective destruction of malignant tissues, with both potentially toxic and beneficial consequences. A pilot clinical trial was undertaken combining tumor necrosis factor-alpha (TNF-alpha) with the monoclonal antibody R24 (MoAb R24) against GD3 ganglioside in patients with metastatic melanoma. Patients received MoAb R24 to recruit leukocytes to the tumor followed by low doses of recombinant TNF-alpha to activate leukocytes. Eight patients were treated and seven patients had mild toxicity. One patient with extensive metastatic melanoma developed tumor lysis syndrome within hours after treatment with almost complete necrosis of bulky tumors in multiple visceral sites. To our knowledge, this is the first documented case of hemorrhagic tumor necrosis in a patient with metastatic cancer in multiple visceral sites.

A marker for neoplastic progression of human melanocytes is a cell surface ectopeptidase.

Adenosine deaminase binding protein (ADAbp) is a cell surface glycoprotein that is expressed by normal melanocytes but not by melanoma, the malignant counterpart. ADAbp is specifically downregulated during malignant transformation of melanocytes. Recently, we have developed a system that progressively transforms melanocytes in vitro in defined steps. Transduction with v-Ha-ras oncogene followed by long-term culture leads to a cell phenotype and genotype that specifically mimics human melanoma. Loss of ADAbp expression occurred concomitantly with the emergence of growth factor independence and appearance of specific chromosomal abnormalities. The cellular function of ADAbp has not been defined. To characterize ADAbp, the mature 110-kD form was purified from human kidney. Five tryptic peptides from purified human ADAbp revealed 100% homology to a serine protease, human dipeptidyl peptidase IV (DPP IV), also known as CD26. DPP IV activity was detected in lysates from human melanocytes and renal carcinoma cells but not melanoma cells, and DPP IV activity could be specifically isolated from melanocytes by binding to ADA or to S27 monoclonal antibody against ADAbp. These findings show that ADAbp is a cell surface ectopeptidase that is tightly regulated during neoplastic transformation of melanocytes.

Molecular cloning and expression of a murine homolog of the human poliovirus receptor gene.

The poliovirus receptor (Pvr) is a member of the immunoglobulin superfamily of proteins, but its function in the cell is not known. Southern blot hybridization analysis indicated that the murine genome contains a sequence homolog of pvr. As a first step toward using the murine pvr homolog (mph) to study the function of Pvr, murine genomic and cDNA clones encoding mph were isolated. mph encodes a polypeptide with extensive sequence similarity to the extracellular domains of the human PVR. mph mRNAs of 2.0 and 3.0 kb are transcribed in the adult mouse brain, the spinal cord, the spleen, the kidney, the heart, and the liver. The Mph protein does not function as a receptor for poliovirus. However, substitution of domain 1 of the Mph protein with the corresponding sequence from pvr produced a chimeric receptor that could bind poliovirus and lead to productive infection. By constructing pvr-mph chimeras, it will be possible to identify the contact points of poliovirus within domain 1 of Pvr. Identification of the ligand and the cellular function of the Mph protein may help us understand the role of Pvr in the cell.

Are we ready for PL 99-457?

Public Law 99-457 has renewed interest in providing preschool education for hearing-impaired children. It is not clear, however, whether we're ready for this mandate to expand services to an ever-younger population and its families. This study presents a "snapshot" of the trends in preschool programs and of the availability of programming to students in various parts of the country. We obtained lists of programs from a sample of state level special education offices, and then sent the programs detailed questionnaires. The returned surveys provided a snapshot of the instructional approaches used in many preschool programs and indicated that there may still be some problems in the availability of preschool programming and in the ability of programs to uniformly provide the sorts of quality comprehensive services mandated by the law.

In vitro studies of 2,4-dihydroxyphenylalanine, a prodrug targeted against malignant melanoma cells.

We have evaluated the chemotherapeutic potential of 2,4-dihydroxyphenylalanine, a targeted prodrug that can be hydroxylated by tyrosinase (monophenol monooxygenase, EC 1.14.18.1) within melanoma cells to form the cellular toxin 2,4,5-trihydroxyphenylalanine (6-hydroxydopa). 2,4-Dihydroxyphenylalanine proved to be cytotoxic to both B-16 and Cloudman melanoma cells in vitro. The immediate effects of 2,4-dihydroxyphenylalanine included inhibition of DNA, RNA, and protein syntheses. In contrast, no decrease in macromolecular synthesis or viability was seen against cultures of MJY-alpha mammary tumor or L-1210 leukemia, two cell types that do not contain tyrosinase. Within the melanoma cultures, greater cytotoxicity was seen against melanotic (tyrosinase-containing) cells than against amelanotic (tyrosinase-lacking) cells. The cytotoxicity of 2,4-dihydroxyphenylalanine was blocked by 1-phenylthiourea, an inhibitor of tyrosinase. These results show that 2,4-dihydroxyphenylalanine is toxic to melanoma cells and that activation of 2,4-dihydroxyphenylalanine requires the presence of tyrosinase.