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 24months. 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

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

Exposure to nickel sulfate (NiSO4) 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 NiSO4 or saline followed by ATP, purinergic receptor antagonists, or saline. We assessed the olfactory epithelium for NiSO4-induced changes using histology and immunohistochemistry 1–7 days postinstillation and compared results to olfactory bulb ablation-induced toxicity. Intranasal instillation of NiSO4 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 NiSO4 evidenced by BrdU incorporation. Treatment with purinergic receptor antagonists significantly reduced NiSO4-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 NiSO4-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

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

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

The release of neurotrophic factor NPY following injury simulation is mediated by a purinergic receptor, phospholipase C, and IP3/IP3 receptor pathway.

<p>(A) P2X_1,7 and P2Y₂ receptors mediate NPY release. Neonatal OE slices from Swiss Webster mice were incubated with vehicle (0.2% DMSO), non-selective P2 purinergic receptor agonist (ATP, 50 µM), P2X_1,7 agonist (BzATP, 50 µM), P2Y_2,4,6 agonist (UTP, 50 µM) or P2X_1,2/3,3 agonist (αβ-MeATP, 50 µM) for 1 hour and the levels of NPY in media were measured by ELISA. *, p<0.05 vs. vehicle (one-way ANOVA followed by Newman-Keuls post-hoc test; n = 6, 4, 4, 4 and 3 replications, respectively.) (B) ATP-induced NPY release is PLC- and IP3 receptor-dependent. Neonatal OE slices from Swiss Webster mice were pre-incubated with vehicle (0.2% DMSO), PLC inhibitor (U73122, 100 µM), or IP3 receptor inhibitor (2-APB, 100 µM) for 45 min prior to addition of vehicle (0.2% DMSO) or ATP (50 µM). Media was collected after 1 hr and the levels of NPY in media were measured by ELISA. *p<0.01 vs. vehicle; #p<0.05 vs. ATP (two-way ANOVA followed by Newman-Keuls post-hoc test; n = 6, 4, 4, 4, 5 and 5 replications, respectively.) (C) IP3 induces NPY release. Neonatal OE slices from Swiss Webster mice were incubated with vehicle (0.09% pluronic acid +0.45% DMSO) or caged iso-IP3/PM (caged IP3, 1.5 µM) and slices were illuminated with unfiltered light from a xenon arc lamp (30 min) to uncage IP3. Media was collected 1 hr later and the levels of NPY were measured by ELISA. p = 0.02 (Student’s t-test; n = 3 and 4 replications.).</p