Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

A series of recombinant herpes simplex virus (HSV-1) vectors have been constructed that encode either the full-length cDNA of the human p75 NGF receptor (p75hNGFR) or truncated forms of the receptor. Infection of cultured fibroblast cells with viral stocks results in abundant expression of all three cDNAs, as detected by affinity cross-linking, immunoblot analysis, and equilibrium binding. Furthermore, viral infection of primary neuronal cultures gives easily detectable p75 expression by immunofluorescence and affinity cross-linking. When p75 was introduced by viral infection into fibroblast cells expressing the trk proto-oncogene, a new binding site was created, consistent with high-affinity NGF binding. This site is not created by the coexpression of truncated forms of p75 that lack either the extracellular ligand binding domain or the cytoplasmic domain of the receptor, suggesting that both of these regions of the receptor are required for the formation of the high-affinity NGF binding site. Hence, these HSV-1 vectors give rise to appropriate NGF receptor binding after viral infection. The application of these HSV-1 constructs to primary neuronal culture and in vivo models of p75NGFR function is discussed.

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 μM. 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 μM 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. © 1996 Wiley‐Liss, Inc.

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