NPY mediates ATP-induced neuroproliferation in adult mouse olfactory epithelium

Abstract

In the CNS, ATP is released upon injury and promotes neuroproliferation via purinergic receptors. In the olfactory epithelium, ATP promotes the synthesis and release of neurotrophic factor NPY in neonates and induces neuroproliferation in neonatal and adult mice. We tested the hypothesis that NPY is involved in ATP-induced neuroproliferation in adult mice olfactory epithelium. Intranasal instillation of ATP significantly increased protein levels and number of NPY⁺ cells. Pre-intranasal instillation of purinergic receptor antagonist PPADS significantly reduced ATP-induced upregulation of NPY. Intranasal instillation of NPY-Y1 receptor antagonist BIBP3226 following ATP instillation significantly inhibited the ATP-induced increase in BrdU incorporation, suggesting that NPY is released after ATP instillation and activates Y1 receptors to promote neuroproliferation. These data indicate that ATP initiates neuroproliferation via NPY upregulation, NPY release, and Y1 receptor activation, and suggests that the olfactory epithelium is good model to study neuroregenerative mechanisms in the CNS.

Introduction

The olfactory epithelium (OE) is a good model to study the mechanisms of neurotrophic factor-regulated neuroregeneration as neurogenesis occurs during embryogenesis and continues throughout life under both physiological conditions and following injury (Graziadei and Monti-Graziadei, 1978). The level of neurogenesis is tightly regulated by a multitude of endogenous positive and negative chemical signals. Chemical, infectious, or traumatic damage upsets the balance of these chemical signals and increases neurogenesis. Although studies suggest that mechanisms regulating embryonic neurogenesis and adult neuroregeneration in the OE may be equivalent (Beites et al., 2005, Murdoch and Roskams, 2007, Schwob, 2002), it is not known if similar regulatory signals mediate both postnatal neurogenesis and adult maintenance. Knowledge of the endogenous factors that stimulate neurogenesis throughout development will advance neuroproliferative replacement therapies aimed at endogenous stem cell recruitment.

One possible positive regulator of neurogenesis is ATP. ATP is released following injury, and could stimulate localized proliferation and regeneration. Indeed, sustained pathologic release of ATP has been observed in spinal cord injury (Wang et al., 2004). In the olfactory system, we previously reported that ATP, via activation of P2 purinergic receptors, increases neuronal precursor cell proliferation and differentiation in neonatal and adult mouse OE (Jia et al., 2009). We found that ATP induces NPY release from neonatal mouse OE slices in vitro (Kanekar et al., 2009), suggesting that NPY may be involved in ATP-induced neuronal proliferation in neonatal mouse OE. NPY is a 36 amino acid peptide reported to induce neuronal precursor proliferation in subventricular zone of the lateral ventricle (Stanic et al., 2008), hippocampus (Ha et al., 2002, Howell et al., 2005) and retina (Milenkovic et al., 2004). In the OE, NPY is expressed in sustentacular cells (Hansel et al., 2001, Kanekar et al., 2009), and a subpopulation of microvillous cells, both of which have processes extending to the basement membrane (Kanekar et al., 2009, Montani et al., 2006), and in the olfactory ensheathing cells that reside in the lamina propria (Ubink et al., 1994). NPY Y1 receptors are expressed in the basal cell layer where the basal progenitor cells are located (Hansel et al., 2001). NPY stimulates proliferation of olfactory neuronal progenitor cells in vitro via Y1 receptor-activated extracellular signal-regulated kinase signaling cascade (Doyle et al., 2008, Hansel et al., 2001). A significant reduction in basal cell proliferation occurs in NPY deficient mice (Hansel et al., 2001) and Y1 receptor knockout mice (Doyle et al., 2008). Collectively, these data indicate that NPY is ideally situated to have a role in promoting basal neuronal progenitor cell proliferation. In the present study, we tested the hypothesis that ATP regulates neurogenesis via NPY in adult mouse OE and compared results across different developmental stages (neonatal vs. adult). We found that ATP activation of P2 purinergic receptors significantly increases NPY protein levels, upregulates NPY expression in the sustentacular and microvillous cells, and induces basal progenitor cell proliferation via activation of NPY Y1 receptors. These data suggest that ATP acts synergistically with other neurotrophic factors, such as NPY, to exert its neuroproliferative action in adult OE. Furthermore, it implies that similar mechanisms and regulatory signals mediate postnatal neurogenesis and adult maintenance.