Chitinase-Like Protein Ym2 (Chil4) Regulates Regeneration of the Olfactory Epithelium via Interaction with Inflammation.

The adult olfactory epithelium (OE) regenerates sensory neurons and nonsensory supporting cells from resident stem cells after injury. How supporting cells contribute to OE regeneration remains largely unknown. In this study, we elucidated a novel role of Ym2 (also known as Chil4 or Chi3l4), a chitinase-like protein expressed in supporting cells, in regulating regeneration of the injured OE in vivo in both male and female mice and cell proliferation/differentiation in OE colonies in vitro We found that Ym2 expression was enhanced in supporting cells after OE injury. Genetic knockdown of Ym2 in supporting cells attenuated recovery of the injured OE, while Ym2 overexpression by lentiviral infection accelerated OE regeneration. Similarly, Ym2 bidirectionally regulated cell proliferation and differentiation in OE colonies. Furthermore, anti-inflammatory treatment reduced Ym2 expression and delayed OE regeneration in vivo and cell proliferation/differentiation in vitro, which were counteracted by Ym2 overexpression. Collectively, this study revealed a novel role of Ym2 in OE regeneration and cell proliferation/differentiation of OE colonies via interaction with inflammatory responses, providing new clues to the function of supporting cells in these processes.SIGNIFICANCE STATEMENT The mammalian olfactory epithelium (OE) is a unique neural tissue that regenerates sensory neurons and nonsensory supporting cells throughout life and postinjury. How supporting cells contribute to this process is not entirely understood. Here we report that OE injury causes upregulation of a chitinase-like protein, Ym2, in supporting cells, which facilitates OE regeneration. Moreover, anti-inflammatory treatment reduces Ym2 expression and delays OE regeneration, which are counteracted by Ym2 overexpression. This study reveals an important role of supporting cells in OE regeneration and provides a critical link between Ym2 and inflammation in this process.

Genetic Ablation of Type III Adenylyl Cyclase Exerts Region-Specific Effects on Cilia Architecture in the Mouse Nose.

We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII), a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE) 1C-/-;PDE4A-/- (simplified as PDEs-/- hereafter) and ACIII-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found), similar to our previous observation in ACIII-/- mice. Remarkably, in a region not included in our previous study, ACIII-/- animals (but not PDEs-/- mice) have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology.

An Olfactory Cilia Pattern in the Mammalian Nose Ensures High Sensitivity to Odors.

In many sensory organs, specialized receptors are strategically arranged to enhance detection sensitivity and acuity. It is unclear whether the olfactory system utilizes a similar organizational scheme to facilitate odor detection. Curiously, olfactory sensory neurons (OSNs) in the mouse nose are differentially stimulated depending on the cell location. We therefore asked whether OSNs in different locations evolve unique structural and/or functional features to optimize odor detection and discrimination. Using immunohistochemistry, computational fluid dynamics modeling, and patch clamp recording, we discovered that OSNs situated in highly stimulated regions have much longer cilia and are more sensitive to odorants than those in weakly stimulated regions. Surprisingly, reduction in neuronal excitability or ablation of the olfactory G protein in OSNs does not alter the cilia length pattern, indicating that neither spontaneous nor odor-evoked activity is required for its establishment. Furthermore, the pattern is evident at birth, maintained into adulthood, and restored following pharmacologically induced degeneration of the olfactory epithelium, suggesting that it is intrinsically programmed. Intriguingly, type III adenylyl cyclase (ACIII), a key protein in olfactory signal transduction and ubiquitous marker for primary cilia, exhibits location-dependent gene expression levels, and genetic ablation of ACIII dramatically alters the cilia pattern. These findings reveal an intrinsically programmed configuration in the nose to ensure high sensitivity to odors.

Muscarinic acetylcholine receptor M3 modulates odorant receptor activity via inhibition of ß-arrestin-2 recruitment.

The olfactory system in rodents serves a critical function in social, reproductive and survival behaviours. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odour-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odours. Here, we show that an M3-R antagonist attenuates odour-induced responses in OSNs from wild-type, but not M3-R-null, mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of ß-arrestin-2 to ORs, resulting in a potentiation of odour-induced responses in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

Activity-dependent modulation of odorant receptor gene expression in the mouse olfactory epithelium.

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.

Postnatal experience modulates functional properties of mouse olfactory sensory neurons.

Early experience considerably modulates the organization and function of all sensory systems. In the mammalian olfactory system, deprivation of the sensory inputs via neonatal, unilateral naris closure has been shown to induce structural, molecular and functional changes from the olfactory epithelium to the olfactory bulb and cortex. However, it remains unknown how early experience shapes the functional properties of individual olfactory sensory neurons (OSNs), the primary odor detectors in the nose. To address this question, we examined the odorant response properties of mouse OSNs in both the closed and open nostril after 4 weeks of unilateral naris closure, with age-matched untreated animals as control. Using a patch-clamp technique on genetically tagged OSNs with defined odorant receptors (ORs), we found that sensory deprivation increased the sensitivity of MOR23 neurons in the closed side, whereas overexposure caused the opposite effect in the open side. We next analyzed the response properties, including rise time, decay time, and adaptation, induced by repeated stimulation in MOR23 and M71 neurons. Even though these two types of neuron showed distinct properties with regard to dynamic range and response kinetics, sensory deprivation significantly slowed down the decay phase of odorant-induced transduction events in both types. Using western blotting and antibody staining, we confirmed the upregulation of several signaling proteins in the closed side as compared with the open side. This study suggests that early experience modulates the functional properties of OSNs, probably by modifying the signal transduction cascade.

Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.

The sense of smell deteriorates in normal aging, but the underling mechanisms are still elusive. Here we investigated age-related alterations in expression patterns of odorant receptor (OR) genes and functional properties of olfactory sensory neurons (OSNs)-2 critical factors that define the odor detection threshold in the olfactory epithelium. Using in situ hybridization for 9 representative OR genes, we compared the cell densities of each OR in coronal nose sections at different ages (3-27 months). The cell density for different ORs peaked at different time points and a decline was observed for 6 of 9 ORs at advanced ages. Using patch clamp recordings, we then examined the odorant responses of individual OSNs coexpressing a defined OR (MOR23) and green fluorescent protein. The MOR23 neurons recorded from aged animals maintained a similar sensitivity and dynamic range in response to the cognate odorant (lyral) as those from younger mice. The results indicate that although the cell densities of OSNs expressing certain types of ORs decline at advanced ages, individual OSNs can retain their sensitivity. The implications of these findings in age-related olfactory deterioration are discussed.

Activity plays a role in eliminating olfactory sensory neurons expressing multiple odorant receptors in the mouse septal organ.

A fundamental belief in the field of olfaction is that each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) type. Here we report that coexpression of multiple receptors in single neurons does occur at a low frequency. This was tested by double in situ hybridization in the septal organ in which greater than 90% of the sensory neurons express one of nine identified ORs. Notably, the coexpression frequency is nearly ten times higher in newborn than in young adult mice, suggesting a reduction of the sensory neurons with multiple ORs during postnatal development. In addition, such reduction is prevented by four-week sensory deprivation or impaired apoptosis. Furthermore, the high coexpression frequency is restored following four-week naris closure performed in young adult mice. The results indicate that activity induced by sensory inputs plays a role in ensuring the one cell-one receptor rule in a subset of olfactory sensory neurons.

Differential development of odorant receptor expression patterns in the olfactory epithelium: a quantitative analysis in the mouse septal organ.

The rodent olfactory epithelium expresses more than 1000 odorant receptors (ORs) with distinct patterns, yet it is unclear how such patterns are established during development. In the current study, we investigated development of the expression patterns of different ORs in the septal organ, a small patch of olfactory epithelium predominantly expressing nine identified ORs. The presumptive septal organ first appears at about embryonic day 16 (E16) and it completely separates from the main olfactory epithelium (MOE) at about postnatal day 7 (P7). Using in situ hybridization, we quantified the densities of the septal organ neurons labeled by specific RNA probes of the nine abundant OR genes from E16 to postnatal 3 months. The results indicate that olfactory sensory neurons (OSNs) expressing different ORs have asynchronous temporal onsets. For instance, MOR256-17 and MOR236-1 cells are present in the septal organ at E16; however, MOR0-2 cells do not appear until P0. In addition, OSNs expressing different ORs show distinct developmental courses and reach their maximum densities at different stages ranging from E16 (e.g. MOR256-17) to 1 month (e.g. MOR256-3 and MOR235-1). Furthermore, early onset does not correlate with high abundance in adult. This study reveals a dynamic composition of the OSNs expressing different ORs in the developing olfactory epithelium.

High-throughput microarray detection of olfactory receptor gene expression in the mouse.

The large number of olfactory receptor genes necessitates high throughput methods to analyze their expression patterns. We have therefore designed a high-density oligonucleotide array containing all known mouse olfactory receptor (OR) and V1R vomeronasal receptor genes. This custom array detected a large number of receptor genes, demonstrating specific expression in the olfactory sensory epithelium for approximately 800 OR genes previously designated as ORs based solely on genomic sequences. The array also enabled us to monitor the spatial and temporal distribution of gene expression for the entire OR family. Interestingly, OR genes showing spatially segregated expression patterns were also segregated on the chromosomes. This correlation between genomic location and spatial expression provides unique insights about the regulation of this large family of genes.

Molecular organization of the olfactory septal organ.

The septal organ in the mammalian nose is a distinct chemosensory organ sitting in the air path. To gain insights into its organization and function, we analyzed the chemoreceptors expressed in this area. By combining cDNA cloning, Affymetrix (Santa Clara, CA) genechips covering all the mouse olfactory receptor genes, and in situ hybridization, we achieved a relatively complete expression profile of the olfactory receptor genes in the septal organ. The majority of the septal neurons express only a few receptor genes in varying patterns, with the top one in approximately 50% of the cells and the top eight together in approximately 93% of the cells. We demonstrated that a single neuron expresses only one receptor by a thorough combination of all the major septal receptor genes in double-labeling studies. These septal receptor genes do not form a single subfamily. Instead, these genes are distributed on a few major branches of the phylogenetic tree covering all the mouse olfactory receptors. Most of these genes are also concentrated in certain areas within the most ventral zone of the main olfactory epithelium, although their expression patterns do not match those in the septal organ. In contrary to the previous view of random distribution, our results indicate that certain olfactory receptors form "hot spots" in the nose.

[Effect of lugu Ganoderma lucidum on low-density lipoprotein oxidation and monocyte adhesion to endothelium].

OBJECTIVE: To study the effect of Lugu Ganoderma Lucidum (LGL) on low-density lipoprotein (LDL) oxidation and monocyte adhesion to endothelium (AdM-E) induced by oxydative LDL and advanced glycosylation endproducts (AGE) by using serum pharmacological technique. METHODS: LDL oxidation was determined by measuring the thiobarbituric acid reactive substances in the supernatants, and AdM-E was determined by measuring myeloperoxidase activity of adherent monocyte. RESULTS: Serum derived from rats 0.5 hrs, 1 hr, 2 hrs, 3 hrs after LGL administering 0.12 g/kg once and 0.5 hrs, 1 hr after LGL administering twice showed no significant effect on LDL oxidation, but the serum from rats 2 hrs, 3 hrs after LGL 0.12 g/kg administering twice or from rats after 10 successive days LGL administering in dose of 0.12 g/kg, 0.24 g/kg and 0.72 g/kg, all could lower the LDL oxidation (P < 0.05). Besides, the serum from rats with 10 days LGL administering of all dosages also could inhibit AdM-E induced by AGE (P < 0.05), and those of 0.24 g/kg and 0.72 g/kg could inhibit AdM-E induced by oxydative LDL (P < 0.05). CONCLUSION: LGL could decrease LDL oxidation and AdM-E induced by AGE or oxydative LDL.