Antibody to the extracellular domain of the low affinity NGF receptor stimulates p75(NGFR)-mediated apoptosis in cultured sympathetic neurons.

Recent evidence has established a role for p75(NGFR) in developmentally regulated neuronal cell death. Although cell death due to NGF withdrawal is a well described, apoptosis in sympathetic neurons through stimulation of p75(NGFR) has not been clearly demonstrated. We have found that an antibody directed against the extracellular domain of murine p75(NGFR) profoundly effects the survival of short-term cultures of sympathetic neurons. Rat superior cervical ganglion neurons grown in the presence of NGF and treated with the bioactive antibody (9651) display a dose-dependent increase in cell death. This effect was independent of NGF concentration and partially reversed by either depolarizing stimuli or forskolin. The response to 9651 seems to act directly through a p75(NGFR)-mediated pathway and not by disturbing p75(NGFR)/TrkA interactions. Moreover, the kinetics of antibody stimulated cell death was more rapid than the cell death resulting from removal of NGF and treatment with CNTF failed to promote neuronal survival in the presence of 9651. Initiation of cell death is often associated with decreased NFkappaB activity, whereas survival or rescue correlates with increased NFkappaB. Increases in NFkappaB, however, have been observed in neurons in several diseases and late in apoptosis in differentiated PC12 cells. Time course studies revealed a rapid decrease in NFkappaB activity and a slight, but persistent increase in binding that correlated with decline in cell numbers 3 hr after treatment. These results suggest the cell death program is initiated shortly after antibody activation of p75(NGFR) and a subpopulation of cells may remain susceptible to rescue.

Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot-Marie-Tooth disease.

CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.

Competition for DNA steroid response elements as a possible mechanism for neuroendocrine integration.

For the analysis of a simple steroid-dependent mating behavior, careful response definition, complete neural circuit delineation and placement of estrogen-responsive cells within this circuit have been accomplished. Molecular studies of two relevant genes have emphasized DNA/RNA hybridization assays and DNA binding techniques. For both the rat preproenkephalin gene and the gene for the progesterone receptor, a strong induction by estrogen, tissue specificity of expression and a sex difference in regulation are prominent phenomena. On the rat preproenkephalin promoter, estrogen (ER) and thyroid receptors may compete for a DNA binding site. Likewise, progesterone (PR) and glucocorticoid receptors may compete for the same sites. On the rat PR gene, interactions between ER and AP-1 binding proteins are of special interest. Such interactions could underlay competitions and synergies between steroid hormones and neurally signalled events in the environment.

Cytokine-induced programmed death of cultured sympathetic neurons.

Programmed cell death (PCD) of sympathetic neurons is inhibited by nerve growth factor. However, factors that induce PCD of these cells are unknown. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, neuropoietic cytokines known to regulate sympathetic neuron gene expression, were examined for effects on survival of cultured sympathetic neurons. Treatment with LIF or ciliary neurotrophic factor caused neuronal death in a dose-dependent fashion. Inhibition of RNA or protein synthesis, or treatment with potassium, all of which prevent PCD after nerve growth factor deprivation, prevented LIF-induced death. The morphologic and ultrastructural characteristics of the neuronal death induced by LIF and by nerve growth factor deprivation were similar. Furthermore, LIF treatment resulted in DNA fragmentation with a characteristic "ladder" on Southern blot analysis. These observations suggest that neuron numbers may be regulated by factors which initiate PCD, as well as by factors which prevent it.