Colleen Cosgrove Hegg

Calcium store-mediated signaling in sustentacular cells of the mouse olfactory epithelium.

Sustentacular cells have structural features that allude to functions of secretion, absorption, phagocytosis, maintenance of extracellular ionic gradients, metabolism of noxious chemicals, and regulation of cell turnover. We present data detailing their dynamic activity. We show, using a mouse olfactory epithelium slice model, that sustentacular cells are capable of generating two types of calcium signals: intercellular calcium waves where elevations in intracellular calcium propagate between neighboring cells, and intracellular calcium oscillations consisting of repetitive elevations in intracellular calcium confined to single cells. Sustentacular cells exhibited rapid, robust increases in intracellular calcium in response to G‐protein coupled muscarinic and purinergic receptor stimulation. In a subpopulation of sustentacular cells, oscillatory calcium transients were evoked. We pharmacologically characterized the properties of purinergic‐evoked increases in intracellular calcium. Calcium transients were elicited by release from intracellular stores and were not dependent on extracellular calcium. BAPTA‐AM, a cytosolic calcium chelator, and cyclopiazonic acid, an endoplasmic reticulum Ca2+‐ATPase inhibitor irreversibly blocked the purinergic‐induced calcium transient. Phospholipase C antagonist U73122 inhibited the purinergic‐evoked calcium transient. 2‐Aminoethoxydiphenyl borate, an inositol‐1,4,5‐trisphosphate (IP3) receptor antagonist, and the ryanodine receptor (RyR) antagonists tetracaine and ryanodine, inhibited the UTP‐induced calcium transients. Collectively, these data suggest that activation of the phospholipase C pathway, IP3‐mediated calcium release, and subsequent calcium‐induced‐calcium release is involved in ATP‐elicited increases in intracellular calcium. Our findings indicate that sustentacular cells are not static support cells, and, like glia in the central nervous system, have complex calcium signaling.

NPY mediates ATP-induced neuroproliferation in adult mouse olfactory epithelium.

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.

Microvillous cells expressing IP3 receptor type 3 in the olfactory epithelium of mice.

Microvillous cells of the main olfactory epithelium have been described variously as primary olfactory neurons, secondary chemosensory cells or non‐sensory cells. Here we generated an IP3R3tm1(tauGFP) mouse in which the coding region for a fusion protein of tau and green fluorescent protein replaces the first exon of the Itpr3 gene. We provide immunohistochemical and functional characterization of the cells expressing IP3 receptor type 3 in the olfactory epithelium. These cells bear microvilli at their apex, and we therefore termed them IP3R3 MV cells. The cell body of these IP3R3 MV cells lies in the upper third of the main olfactory epithelium; a long thick basal process projects towards the base of the epithelium without penetrating the basal lamina. Retrograde labeling and unilateral bulbectomy corroborated that these IP3R3 MV cells do not extend axons to the olfactory bulb and therefore are not olfactory sensory neurons. The immunohistochemical features of IP3R3 MV cells varied, suggesting either developmental stages or the existence of subsets of these cells. Thus, for example, subsets of the IP3R3 MV cells make contact with substance P fibers or express the purinergic receptor P2X3. In addition, in recordings of intracellular calcium, these cells respond to ATP and substance P as well as to a variety of odors. The characterization of IP3R3 MV cells as non‐neuronal chemoresponsive cells helps to explain the differing descriptions of microvillous cells in the literature.

Purinergic receptor activation evokes neurotrophic factor neuropeptide Y release from neonatal mouse olfactory epithelial slices.

One premise regarding the mechanism of injury‐evoked neuroregeneration is that injured cells induce the release of neurotrophic factors to trigger neurogenesis. Extracellular purine nucleotides exert multiple neurotrophic actions in the central nervous system mediated via activation of purinergic receptors. However, whether purinergics have a neurotrophic role in the olfactory neuroepithelium has not been investigated. Thus, we monitored the ATP‐induced release of neuropeptide Y (NPY), a neuropeptide that increases neuroproliferation in the olfactory epithelium. To visualize NPY release, slices of olfactory epithelium from neonatal mice were cultured on nitrocellulose paper. Immunoassays of the nitrocellulose demonstrated NPY immunoreactivity in regions corresponding to the olfactory epithelium of the nasal cavity. One hour of exposure to exogenous ATP (100, 500 μM) significantly increased the number of olfactory epithelium slices that released NPY from 25% ± 6% to 60% ± 7% or 71% ± 10% (P = 0.001). The purinergic receptor antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS; 25 μM) and suramin (100 μM) significantly reduced the number of olfactory epithelium slices exhibiting ATP‐evoked NPY release to 18% ± 11% (P = 0.004), indicating that NPY release is mediated by activation of purinergic receptors. Released NPY was quantified by enzyme and radioimmunoassays. Exogenous ATP or UTP significantly increased the amount of NPY released. Overall, this study demonstrates that purinergic receptor activation mediates the release of neurotrophic factor NPY in the olfactory epithelium and provides pharmacological targets to promote regeneration of damaged olfactory epithelium.

Satratoxin G-induced apoptosis in PC-12 neuronal cells is mediated by PKR and caspase independent

Satratoxin G (SG) is a macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum, a mold suggested to play an etiologic role in damp building-related illnesses. Acute intranasal exposure of mice to SG specifically induces apoptosis in olfactory sensory neurons of the nose. The PC-12 rat pheochromocytoma cell model was used to elucidate potential mechanisms of SG-induced neuronal cell death. Agarose gel electrophoresis revealed that exposure to SG at 10 ng/ml or higher for 48-h induced DNA fragmentation characteristic of apoptosis in PC-12 cells. SG-induced apoptosis was confirmed by microscopic morphology, hypodiploid fluorescence and annexin V-fluorescein isothiocyanate (FITC) uptake. Messenger RNA expression of the proapoptotic genes p53, double-stranded RNA-activated protein kinase (PKR), BAX, and caspase-activated DNAse was significantly elevated from 6 to 48 h after SG treatment. SG also induced apoptosis and proapoptotic gene expression in neural growth factor-differentiated PC-12 cells. Although SG-induced caspase-3 activation, caspase inhibition did not impair apoptosis. Moreover, SG induced nuclear translocation of apoptosis-inducing factor (AIF), a known contributor to caspase-independent neuronal cell death. SG-induced apoptosis was not affected by inhibitors of oxidative stress or mitogen-activated protein kinases but was suppressed by the PKR inhibitor C16 and by PKR siRNA transfection. PKR inhibition also blocked SG-induced apoptotic gene expression and AIF translocation but not caspase-3 activation. Taken together, SG-induced apoptosis in PC-12 neuronal cells is mediated by PKR via a caspase-independent pathway possibly involving AIF translocation.

ATP differentially upregulates fibroblast growth factor 2 and transforming growth factor alpha in neonatal and adult mice: effect on neuroproliferation

Multiple neurotrophic factors play a role in proliferation, differentiation and survival in the olfactory epithelium (OE); however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic factors, fibroblast growth factor 2 (FGF2) and transforming growth factor alpha (TGFα), and that these neurotrophic factors have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased 20 h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse olfactory epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult olfactory epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal growth factor receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse olfactory epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development.

Nickel Sulfate Induces Location-Dependent Atrophy of Mouse Olfactory Epithelium: Protective and Proliferative Role of Purinergic Receptor Activation

Exposure to nickel sulfate (NiSO(4)) leads to impaired olfaction and anosmia through an unknown mechanism. We tested the hypothesis that ATP is released following NiSO4-induced injury and that ATP promotes regenerative cell proliferation in the olfactory epithelium (OE). Male Swiss Webster mice were intranasally instilled with NiSO(4) or saline followed by ATP, purinergic receptor antagonists, or saline. We assessed the olfactory epithelium for NiSO(4)-induced changes using histology and immunohistochemistry 1-7 days postinstillation and compared results to olfactory bulb ablation-induced toxicity. Intranasal instillation of NiSO(4) produced a dose- and time-dependent reduction in the thickness of turbinate OE. These reductions were due to sustentacular cell loss, measured by terminal dUTP nick-end labeling (TUNEL) staining at 1-day postinstillation and caspase-3-dependent apoptosis of olfactory sensory neurons at 3 days postinstillation. A significant increase in cell proliferation was observed at 5 and 7 days postinstillation of NiSO(4) evidenced by BrdU incorporation. Treatment with purinergic receptor antagonists significantly reduced NiSO(4)-induced cell proliferation and posttreatment with ATP significantly increased cell proliferation. Furthermore, posttreatment with ATP had no effect on sustentacular cell viability but significantly reduced caspase-3-dependent neuronal apoptosis. In a bulbectomy-induced model of apoptosis, exogenous ATP produced a significant increase in cell proliferation that was not affected by purinergic receptor antagonists, suggesting that ATP is not released during bulbectomy-induced apoptosis. ATP is released following NiSO(4)-induced apoptosis and has neuroproliferative and neuroprotective functions. These data provide therapeutic strategies to alleviate or cure the loss of olfactory function associated with exposure to nickel compounds.

An IP3R3- and NPY-expressing microvillous cell mediates tissue homeostasis and regeneration in the mouse olfactory epithelium.

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3+/−, and IP3R3−/− mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3−/− mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3−/− mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3−/− mice. The reductions in both NPY release and number of progenitor cells in IP3R3−/− mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.

Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium.

BackgroundATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices.ResultsUnder control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone.ConclusionsThe constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.

Effect of IP3R3 and NPY on age-related declines in olfactory stem cell proliferation

Losing the sense of smell because of aging compromises health and quality of life. In the mouse olfactory epithelium, aging reduces the capacity for tissue homeostasis and regeneration. The microvillous cell subtype that expresses both inositol trisphosphate receptor type 3 (IP3R3) and the neuroproliferative factor neuropeptide Y (NPY) is critical for regulation of homeostasis, yet its role in aging is undefined. We hypothesized that an age-related decline in IP3R3 expression and NPY signaling underlie age-related homeostatic changes and olfactory dysfunction. We found a decrease in IP3R3(+) and NPY(+) microvillous cell numbers and NPY protein and a reduced sensitivity to NPY-mediated proliferation over 24 months. However, in IP3R3-deficient mice, there was no further age-related reduction in cell numbers, proliferation, or olfactory function compared with wild type. The proliferative response was impaired in aged IP3R3-deficient mice when injury was caused by satratoxin G, which induces IP3R3-mediated NPY release, but not by bulbectomy, which does not evoke NPY release. These data identify IP3R3 and NPY signaling as targets for improving recovery following olfactotoxicant exposure.