Wheatley, Susan Caroline; (1992) Characterisation of novel neuronal cell lines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Novel hybrid neuronal cell lines, derived from neonatal rat primary dorsal root ganglion neurons and a neuroblastoma, have been studied to ascertain the similarity of some of their properties to those of primary sensory neurons. Particular attention has been paid to the non-permissive interaction between these cells and Herpes Simplex Virus, as the natural site of HSV latent infections is the sensory ganglion. The permissivity of the ND cells has therefore been established with respect to a permissive ceil line. The mechanism of this non-permissive interaction has also been investigated. Elements from the HSV IE gene promoters that bind the HSV IE gene transcription activating complex containing oct-1 and VP16, are shown to also bind an inhibitor of viral IE gene transcription. Such sequences, when introduced into a cell containing IE genes, are able to increase the level of transcription of the IE genes that is normally low. A model of transcriptional inhibition of the viral immediate-early genes in neuronal cells is proposed. The effect on HSV IE gene expression of cyclic AMP is shown to be either stimulatory or repressive, depending on the concentration. It is shown that the transcription of the IE genes is stimulated at low levels of cyclic AMP via a cyclic AMP responsive element in the IE-1 gene promoter, which is likely to be responsible for the subsequent induction of at least IE-3 gene transcription. This may represent a molecular mechanism of reactivation from latent HSV infections, particularly as cyclic AMP has been observed by others to reactivate latent HSV infections. ND cells can be differentiated in vitro by modulation of the culture medium. Little change in gene expression occurs during the morphological differentiation, suggesting that ND cells are mature in the undifferentiated form. However, the pathways of exocytosis mature as neurites are extended and this is demonstrated by a redistribution of proteins associated with exocytotic vesicles to the neurite tips. The ND cells represent many aspects of the dorsal root ganglion neuron whilst maintaining the immortal phenotype from the neuroblastoma. These cells therefore represent an in vitro model of the cells of dorsal root ganglion as they exhibit a non-permissive interaction with HSV and on differentiation acquire the pathways of regulated exocytosis.

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