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

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

IP3R3^−/− mice have fewer basal cells but a normal rate of proliferation in the OE.

<p>(<b>A–L</b>) Representative immunoreactivity to cellular markers in adult C57BL/6 (A, C, E, G, I, K) and IP3R3^−/− (B, D, F, H, J, L) mice: (A–B) horizontal basal cell marker cytokeratin 5 (CK5), (C–D) proneural transcription factor MASH1, (E–F) immature neuronal marker GAP43, (G–H) mature neuronal marker OMP, (I–L) proliferation cell marker PCNA and BrdU. DAPI (blue) demarcates the nuclei. Scale bar, 10 µm, shown in A is relevant for A–I. (<b>M</b>) The number of CK5⁺ HBCs, MASH1⁺ progenitor cells and GAP43⁺ immature neurons in the OE of IP3R3^−/− mice are significantly reduced. *, p<0.01 vs. respective control in C57BL/6 (Student’s t-test for each cell marker; n = 9–12 sections from 3–4 mice. Refer to legend in (O) for (M–O). (<b>N</b>) The number of OMP⁺ neurons in the OE of IP3R3^−/− is similar to C57BL/6 mice (p = 0.39, Student’s t-test; n = 9–12 sections from 3–4 mice.) (<b>O</b>) The rate of proliferation measured by PCNA expression and BrdU incorporation is comparable in the OE of IP3R3^−/−, IP3R3^+/− and C57BL/6 mice. p>0.05, Student’s t-test and one way ANOVA, respectively; n = 9–12 sections from 3–4 mice. Legend refers to M–O.</p