Faculty Bibliography

The generation of different glial cell types in the central nervous system depends upon a wide variety of proliferative and differentiative signals. Here we report that changes in the levels of cyclin-dependent kinase 2 (CDK2) and the cell cycle inhibitor p27kip1 accompany the differentiation of central glia-4 (CG-4) progenitor cells to an astrocytic cell phenotype in the presence of fetal calf serum. Although a decrease in CDK2 levels was observed in both oligodendrocyte and astrocyte cells derived from CG-4 cells, a striking increase in the levels of p27 was observed during the differentiation of astrocyte cells. In astrocyte cell extracts, inhibition of CDK2 activity could be overcome with exogenously added cyclin E. Furthermore, depletion of p27 from astrocyte extracts lowered the amount of cyclin E required for CDK2 activation. Taken together, these results suggest that the inhibitory action of p27 upon cyclin E-CDK2 may prevent entry of cells into the S phase and regulate the progression of CG-4 cells toward an astrocytic lineage

Although ligand-induced dimerization or oligomerization of receptors is a well established mechanism of growth factor signaling, increasing evidence indicates that biological responses are often mediated by receptor trans-signaling mechanisms involving two or more receptor systems. These include G protein-coupled receptors, cytokine, growth factor and trophic factor receptors. Greater responsiveness and inhibitory signaling responses are provided when different signaling pathways merge through receptor trans-signaling

Members of the nerve growth factor (NGF) family promote the survival of neurons during development. NGF specifically activates the receptor trkA, initiating a signal transduction cascade which ultimately blocks cell death. Here we show that NGF can have the opposite effect, inducing the death of mature oligodendrocytes cultured from postnatal rat cerebral cortex. This effect was highly specific, because NGF had no effect on oligodendrocyte precursors and astrocytes. Other neurotrophins such as brain-derived neurotrophin factor (BDNF) and neurotrophin-3 (NT-3) did not induce cell death. NGF binding to mature oligodendrocytes expressing the p75 neurotrophin receptor, but not trkA, resulted in a sustained increase of intracellular ceramide and c-Jun amino-terminal kinase (JNK) activity, which are thought to participate in a signal transduction pathway leading to cell death. Taken together, these results indicate that NGF has the ability to promote cell death in specific cell types through a ligand-dependent signalling mechanism involving the p75 neurotrophin receptor

Precursor cells found in the subventricular zone (SVZ) of the adult brain can undergo cell division and migrate long distances before differentiating into mature neurons. We have investigated the possibility of introducing genes stably into this population of cells. Replication-defective adenoviruses were injected into the SVZ of the lateral ventricle of adult mice. The adenoviruses carried a cDNA for the LacZ reporter or the human p75 neurotrophin receptor, for which species-specific antibodies are available. Injection of the viruses into the SVZ led to efficient labeling of neuronal precursors. Two months after viral injection, infected cells were detected in the olfactory bulb, a significant distance from the site of injection. Labeled periglomerular and granular neurons with extensive dendritic arborization were found in the olfactory bulb. These results demonstrate that foreign genes can be efficiently introduced into neuronal precursor cells. Furthermore, adenovirus-directed infection can lead to long-term stable gene expression in progenitor cells found in the adult central nervous system

The low affinity p75 neurotrophin receptor (p75NTR) is a cysteine-rich transmembrane glycoprotein which is frequently overexpressed in advanced stages of human melanoma. The biological consequences of this overexpression are unknown; however, it has recently been shown that p75NTR can enhance the invasive potential of melanoma cells in vitro. In the present study we examined cell lines established from normal human melanocytes and metastatic melanomas for expression of p75NTR mRNA and protein. The results showed that, compared with normal melanocytes, levels of p75NTR-specific protein were high in seven melanoma lines, markedly decreased in two melanoma lines and comparable in two melanoma lines. The conserved transmembrane domain of p75NTR was analysed for point mutations by single strand conformation polymorphism analysis and direct DNA sequencing. Identical point mutations were detected in the transmembrane domain of p75NTR in the two melanoma lines with reduced p75NTR protein expression, which resulted in the substitution of the uncharged amino acid Gly for the negatively-charged Asp

The mouse Fas/APO-1 antigen represents a 45-kilodalton transmembrane receptor that initiates apoptosis by a poorly defined signaling mechanism. The cytoplasmic domain of Fas does not display any known enzymatic activities but is capable of interacting with a number of proteins that were identified recently using the yeast interactive cloning method. To investigate direct biochemical interactions from cellular lysates prepared from Fas-responsive cells, a series of recombinant glutathione S-transferase-mouse Fas fusion proteins representing different regions of the mouse Fas cytoplasmic domain was used. Polypeptides of 25, 50, and 70 kilodaltons were found to associate with the Fas intracellular domain, and this binding was stable in the presence of 1 M NaCl. These interactions were also detected using a mouse Fas fusion protein containing an Ile to Asn mutation, which is responsible for a lymphoproliferative disorder in certain strains of mice (lprcg). Furthermore, the binding of cellular proteins to Fas could be blocked upon incubation with a polyclonal antibody directed against the cytoplasmic domain of Fas. The strong association of cellular proteins with the cytoplasmic region implies that constitutive interactions may exist to regulate apoptotic signaling through the Fas antigen

Ceramide is a lipid second messenger implicated in the mechanism of apoptotic cell death. The effect of the cell-permeable ceramide analogue C2 has been tested on primary cortical cultures of neurons, astrocytes, and oligodendrocytes as well as on the bipotential glial precursor cell line CG-4. After 24 hr of treatment, C2 ceramide induced a dose-dependent cell death in primary oligodendrocytes and precursor cells, with a maximum effect at 10 microM. Commitment of oligodendrocytes to cell death occurred within the first 6 hr of treatment. Ultramicroscopic analysis of primary oligodendrocytes exposed to C2 ceramide for 3.5 hr revealed extensive membrane blebbing in the absence of nuclear condensation. In contrast, similar treatment of primary neuronal or astrocytic cortical cultures had no effect on cell survival. Neurons and astrocytes were resistant to 10 microM C2 ceramide. Furthermore, bipotential progenitors that were differentiated toward astrocytes also became resistant to ceramide treatment as they acquired a mature astrocytic phenotype. These experiments suggest that cell type specific factors are required for ceramide-mediated cell death in the nervous system

Five decades of research on NGF have led to the discovery of a small family of evolutionarily conserved proteins, which have vital functions in the survival and neuronal development of specific neuronal populations. The generation of mice lacking neurotrophin expression has recapitulated classic experiments using anti-NGF antibodies to dissect the physiological effects of trophic factor deprivation (73). Very similar outcomes resulted from both the NGF immunodepletion experiments and the transgenic mouse experiments. The genetic results also verify the structural predictions made from binding results in heterologous cells. The findings in cell culture and animal experiments clearly indicate the efficacy of neurotrophic factors for promoting the survival of prominent neuronal populations such as sensory and motor neurons. The high degree of conservation of neurotrophin structure is accompanied by a surprising variation in the amino acid contacts used by each neurotrophin with p75 and the trk receptor family members. It is this variation that may provide specificity for each ligand-receptor complex. The future challenge will be to make use of this knowledge to design effective therapeutic strategies to treat neurodegeneration and nerve injury

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of helix-loop-helix (HLH) proteins during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor. Gel mobility shift assays using PC12 cell nuclear extracts demonstrated that active basic HLH complexes exist throughout differentiation. Addition of exogeneous Id1 protein, a negative regulator of basic HLH proteins, disrupted specific complexes formed by PC12 cell nuclear extracts on a CANNTG consensus oligonucleotide. To identify possible novel basic HLH proteins in these complexes, a glutathione S-transferase-Id1 fusion protein was used to screen a PC12 cell cDNA expression library. A single clone representing the rat E2-2 gene was identified. Sequential immunoprecipitations with antibodies to each HLH protein revealed an association between Id1 and E2-2 that could be detected in both untreated and nerve growth factor-treated PC12 cell lysates. These experiments define a new HLH interaction between Id1 and E2-2 in neuronal cells and suggest that neuronal differentiation may be regulated by HLH proteins in a distinctive manner

The neurotrophin family of survival factors is distinguished by a unique receptor-signaling system that is composed of two transmembrane receptor proteins. Nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and NT-4/5 share similar protein structures and biological functions and interact with two different types of cell-surface proteins, the Trk family of receptor tyrosine kinases, and the p75, or low-affinity neurotrophin receptor. An important question is whether a dual receptor system is necessary for neurotrophin action. Evidence indicates that co-expression of the two genes for the p75 receptor and the Trk NGF receptor can potentially lead to greater responsiveness to NGF, and suggests additional levels of regulation for the family of neurotrophin factors