Nitric oxide mediates the neuroproliferative effect of Neuropeptide Y

Abstract

Neuropeptide Y (NPY) is widely expressed in both the central and peripheral nervous system and has an important role in the regulation of adult hippocampal neurogenesis by mediating the proliferation of neural precursor cells in both health and disease. The mechanisms underlying this neuroproliferative effect of NPY, however, are unknown. The aim of this project was to investigate these cellular pathways and the possible involvement of nitric oxide (NO) in NPY-mediated neuroproliferation using postnatal rat hippocampal cultures in vitro. NPY was found to have a purely proliferative effect on hippocampal neural precursor cells. The role of NO was explored by inhibiting the NO synthesising enzyme, nitric oxide synthase (NOS), which abolished the proliferative effect of NPY and supported the involvement of NO in NPY-mediated proliferation. Pharmacological analyses using subtype-selective inhibitors suggested that the neuronal isoform of NOS is the sole NOS subtype involved, which was expressed by both nestin+ precursors and class III ?-tubulin+ neurons, the cell types previously shown to be responsive to NPY. The involvement of NO was further verified through loading hippocampal cells with an NO indicator, diaminofluorescein diacetate, where an increase in NO/N2O3 production was observed in nestin+ precursors and class III ?-tubulin+ neurons in response to NPY treatment. The downstream signalling pathways coupling NPY-mediated NO synthesis to cell proliferation were identified, through the use of selective pharmacological agonists and antagonists, as soluble guanylate cyclase, cGMP-dependent protein kinase (PKG) and the extracellular signal-regulated kinases (ERK) 1/2. By assessing levels of NPY-mediated ERK 1/2 phosphorylation in response to NOS inhibition, it was found that ERK 1/2 activation was mediated only via NOS/NO mechanisms. This proliferative cGMP-PKG-ERK 1/2 signalling cascade appears to be mediated by intracellularly released NO, while on the other hand, the addition of extracellular NO through the application of NO donors exerted an inhibitory effect on neural precursor cell proliferation. In addition to demonstrating the dual nature of NO, this is the first time that the signalling mechanisms underlying the proliferative effect of NPY on neural precursor cells have been described. Understanding the mechanisms underlying the proliferation of neural precursor cells will ultimately be beneficial by allowing the development of novel therapeutic interventions for promoting hippocampal neurogenesis.
To analyse the role of NO in the NPY-mediated neuroproliferation of hippocampal cells in three-dimensional (3D) cultures, Laponite, a novel synthetic silica hydrogel, was used. Culture medium-based Laponite hydrogels were developed before cell viability within the hydrogels were assessed by culturing hippocampal monolayers under gel cover. Hydrogel cover, however, resulted in cell behaviour reminiscent of preservation/fixation as monolayers showed no spatial or morphological changes over time, with one possible explanation being the high gel osmolarity. Although attempts at cell seeding showed more positive results, with cells adhering to a low heavy metal content variation of the hydrogel, determination of cell viability remained a problem due to prominent dye-gel binding. Although the rheological properties of Laponite make its use attractive, the biocompatibility of the hydrogels with hippocampal cells still require further optimisation if they are to be used as cell culture matrices.

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Identifiers

ORCID for Philip L. Newland: orcid.org/0000-0003-4124-8507

ORCID for George Attard: orcid.org/0000-0001-8304-0742

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