Hu-Tsai, Mi-i; (1994) The role of NGF in the control of chemical phenotype and growth of adult primary sensory neurons. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Neuronal development and maintenance of function in the adult nervous system are both influenced by soluble trophic factors derived from target tissues. Nerve growth factor (NGF), is the prototypic growth factor that defines the properties and functions of the neurotrophins. It regulates survival, neurite growth, and the chemical phenotype of certain neuronal populations including sympathetic neurons, a subpopulation of primary sensory neurons, and basal forebrain cholinergic neurons. I have investigated the role of NGF in the control of the chemical phenotype of sensory neurons by studing capsaicin sensitivity, whose expression is dependent on the presence of NGF. A retrograde labelling technique combined with a cobalt uptake assay in cultured DRG neurons has been used to study the distribution of capsaicin sensitivity in relation to different peripheral targets. I have found regional differences between skin, skeletal muscle and bladder with respect to capsaicin- sensitive afferent innervation. I have also looked at the expression of a growth related protein: GAP-43 and neurite outgrowth in relation to NGF. NGF promotes neurite outgrowth in cultured DRG cells and regulates GAP-43 mRNA levels in PC 12 cells. In order to determine whether NGF modifies GAP-43 expression in adult neurons, a technique using non-isotopic in situ hybridization with an alkaline phosphatase-linked oligo probe was undertaken on cultured DRG neurons. No difference in the expression of GAP-43 mRNA was found between neurons cultured in the presence or absence of NGF. In contrast, cultures of neurons that had been pre-axotomized showed substantial increases in GAP-43 gene expression. NGF did increase neurite outgrowth compared to cells cultured in the presence of anti-NGF, but this effect was substantially less than that resulting from pre-axotomy, which initiated an early and profuse neurite outgrowth. To study the distribution of the high affinity NGF receptor, I have raised antibodies (anti-peptide trk 1, 2 and 3) to 3 distinct peptide sequences of the human p140trk no homology to trkB or trkC, but with partial homology with rat pl40trk. These antibodies recognize a 140-150k protein from extracts of PC12 cells and rat cerebellum. In addition, a distinct immunocytochemical staining pattern was found for these antibodies. The majority of adult DRG, nodose and SCG neurons are immunoreactive. In the CNS high levels of staining are found in motoneuron pools, hippocampal cells and Purkinje cells in the cerebellum. In situ hybridization using oligonucleotide probe complementary to the mRNA of peptide trk 1 cDNA sequences is in general agreement with the immunocytochemical results. This distribution is not fully congruent with the distribution of NGF responsive cells, NGF high affinity binding sites or in situ hybridization for p140trk mRNA. Cross-linking of [125I]NGF to p140trk and immunoblotting has shown that the novel antigen is not rat p140trk, nor its isoform. Neverless, the 140-150k molecular weight of the antigen, a size similar to those of the trk family of tyrosine protein kinase and the sequence homology to p140trk, together with the similarities to trkB and trkC distribution patterns in the central nervous system suggests that the novel antigen is a novel member of or closely related to the trk family of transmembrane proteins.

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