Regulation of VIP and other neuropeptides by c-Jun in sensory neurons: implications for the neuropeptide response to axotomy.

Peripheral axotomy of adult rat sensory neurons causes induction of the transcription factor c-Jun and increased expression of the neuropeptides vasoactive intestinal polypeptide (VIP), galanin and neuropeptide Y. To determine whether VIP induction is dependent on transcriptional regulation by c-Jun, we exploited the fact that c-Jun and VIP are also induced in cultured sensory neurons. We blocked c-Jun synthesis by microinjecting antisense oligonucleotides and found that VIP expression, determined by quantitative immunofluorescence, was specifically reduced.

Intracellular Microinjection: A Powerful Tool in the Study of Neuronal Gene Expression and Function.

Neurons in primary culture provide a useful experimental system with which to explore the functions of neuronal genes and the mechanisms regulating their expression. Both types of study call for transfection of neurons with plasmid DNA constructs that express either the gene of interest or a reporter gene under the transcriptional control of sequences from the gene being studied. However, the phenotypic heterogeneity and limited cell numbers that are often a feature of primary neuronal culture preparations mean that methods commonly used for bulk transfection and analysis of cell line cultures are inappropriate or difficult to apply.

Repression of preprotachykinin-A promoter activity is mediated by a proximal promoter element.

The rat preprotachykinin-A promoter, which is able to direct reporter gene expression in adult dorsal root ganglia neurons grown in culture, has no detectable activity in HeLa and PC12 cells. DNAase 1 footprinting and electrophoretic mobility shift analyses with HeLa nuclear extract indicated the presence of a protein complex binding to a region of the rat preprotachykinn-A gene promoter between the TATA box and the major transcriptional start site. We demonstrate that the sequence of the preprotachykinin-A promoter spanning nucleotides -47 to +92 functions to repress reporter gene expression in HeLa and PC12 cells but not in adult rat dorsal root ganglia grown in culture, and that this repression is correlated with a protein(s) binding to the element between the TATA box and major transcription initiation site.

An activator element within the preprotachykinin-A promoter.

The rat Preprotachykinin-A promoter (PPT) directs high levels of expression in dorsal root ganglia (DRG) neurons in culture either endogenously or when linked to a receptor construct. It is not active in any of the established tissue culture cell lines which we have analyzed. To search for transcriptional regulators within this promoter we have started to dissect the promoter into individual elements to determine their function.

Neuropeptide expression by newborn and adult rat sensory neurons in culture: effects of nerve growth factor and other neurotrophic factors.

Adult rat dorsal root ganglion sensory neurons in culture require nerve growth factor for synthesis of substance P and calcitonin gene-related peptide but express vasoactive intestinal peptide independently of nerve growth factor. In contrast, the same neurons from newborn rats do not express detectable vasoactive intestinal polypeptide when cultured with nerve growth factor. To further explore the mechanisms regulating neuropeptide expression in these cells, I compared the effects of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor and leukaemia inhibitory factor on substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and somatostatin expression in rat dorsal root ganglion cultures.