Cellular and molecular characterization of oxidative stress in olfactory epithelium of Harlequin mutant mouse.

Oxidative stress in the olfactory system is a major factor associated with age-related olfactory impairment, although the mechanisms by which this occurs are not completely understood. The Harlequin mutant mouse (Hq/Y), which carries an X-linked recessive mutation in the Aifm1 gene, is a model of progressive oxidative stress-induced neurodegeneration in the cerebellum and retina. To determine whether the Hq/Y mutant mouse is a suitable model of oxidative stress-associated olfactory aging, we investigated cellular and molecular changes in the olfactory epithelium (OE) and olfactory bulb (OB) of 6-month-old male Hq/Y mice compared to those in sex-matched littermate controls (+/Y) and in age- and sex-matched C57BL/6 mice. Immunoreactivity for apoptosis-inducing factor, the protein product of Aifm1, was localized in mature olfactory sensory neurons (mOSNs) in +/Y mice but was rarely detected in Hq/Y mice. Hq/Y mice also exhibited increased lipofuscin autofluorescence and increased immunoreactivity for an oxidative DNA/RNA damage marker in mOSNs and in mitral/tufted cells in the OB and an increased number of cleaved caspase-3 immunoreactive apoptotic cells in the OE. Microarray analysis demonstrated that Aifm1 expression was down-regulated by 80% in the OE of Hq/Y mice compared to that in +/Y mice. Most significantly, regulated genes were classified into functional categories of cell signaling/apoptosis/cell cycle, oxidative stress/aging, and cytoskeleton/extracellular matrix/transport-associated. Analysis with EASE software indicated that the functional categories significantly overrepresented in Hq/Y mice included up-regulated mitochondrial genes and down-regulated cytoskeletal organization- and neurogenesis-related genes. Our results strongly support the Hq/Y mutant mouse being a novel model for mechanistic studies of oxidative stress-associated olfactory aging.

Molecular mechanisms underlying anti-inflammatory phenotype of neonatal splenic macrophages.

Neonatal humans and rodents are susceptible to infection with encapsulated bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines by splenic macrophages (MPhi) from neonates. In this study, we show that when stimulated with a variety of agonists to TLR2, -4, and -9, neonatal MPhi make less proinflammatory cytokines and more IL-10 than adult MPhi. IL-10 appears to have a role in the decreased proinflammatory cytokine production, as neonatal MPhi treated with anti-IL-10 receptor antibody or from IL-10(-/-) mice produced levels of proinflammatory cytokines at a level comparable with that produced by adult MPhi. A microarray analysis of RNA from resting and LPS-stimulated MPhi from neonatal and adult mice showed that expression of a large number of genes encoding cytokines, chemokines, and their receptors was decreased dramatically in the neonatal MPhi, although some cytokines, including IL-10 and IL-16, were enhanced. Several genes in the TLR signaling pathway leading to NF-kappaB activation were down-regulated, which may account for the decreased chemokine and cytokine synthesis. It is surprising that p38alpha MAPK, known to be required for TLR-induced cytokine secretion, was enhanced in the neonatal MPhi. Our studies with the p38 MAPK inhibitor SB203580 suggested that excess p38 MAPK activity can be inhibitory for TLR2-, -4-, and -9-induced production of proinflammatory cytokines but not IL-10. The anti-inflammatory phenotype of the neonatal Mphi may be unique to the developing organism, although it compromises the neonate's ability to respond to encapsulated bacteria.

Macrophage depletion in the murine olfactory epithelium leads to increased neuronal death and decreased neurogenesis.

Apoptosis of olfactory sensory neurons (OSNs) induced by olfactory bulbectomy (OBX) leads to the activation of resident macrophages within the olfactory epithelium (OE). These macrophages phagocytose degenerating OSNs and secrete chemokines, which recruit additional macrophages into the OE, and cytokines/growth factors, which regulate basal cell proliferation and differentiation and maturation of OSNs. In this study we apply for the first time the use of liposome-encapsulated clodronate to selectively deplete macrophages during the OSN degeneration/regeneration cycle in order to elucidate the role(s) of macrophages in regulating cellular mechanisms that lead to apoptosis and neurogenesis. Mice were injected intranasally and intravenously with either liposome-encapsulated clodronate or empty liposomes prior to and after OBX or sham OBX. At 48 hours after surgery the numbers of macrophages in the OE of both sham and OBX clodronate-treated mice were significantly reduced compared to liposome-treated controls (38% and 35%, respectively, P < 0.05). The reduction in macrophage numbers was accompanied by significant decreases in OE thickness (22% and 21%, P < 0.05), the number of mOSNs (1.2- and 1.9-fold, P < 0.05), and basal cell proliferation (7.6- and 3.8-fold, P < 0.005) in sham and OBX mice, respectively, compared to liposome-treated controls. In OBX mice there was also increased immunoreactivity for active caspase-3 in the OE and olfactory nerves of clodronate-treated OBX mice compared to liposome-treated controls. These results indicate that macrophages modulate the OSN population in the normal and target-ablated murine OE by influencing neuronal survival and basal cell proliferation, resulting in neurogenesis and replacement of mature OSNs.

Oxidative stress in the aging murine olfactory bulb: redox proteomics and cellular localization.

A recent proteomics analysis from our laboratory demonstrated that several oxidative stress response proteins showed significant changes in steady-state levels in olfactory bulbs (OBs) of 20- vs. 1.5-month-old mice. Oxidative stress may result in protein oxidation. In this study, we investigated two forms of protein oxidative modification in murine OBs: carbonylation and nitration. Redox proteomics with two-dimensional gel electrophoresis, Western blotting, protein digestion, and mass spectrometry was used to quantify total and specific protein carbonylation and to identify differentially carbonylated proteins and determine the carbonylation status of previously identified proteins in OBs of 1.5- and 20-month-old mice. Immunohistochemistry was used to demonstrate the relative intensity and localization of protein nitration in OBs of 1.5-, 6-, and 20-month-old mice. Total protein carbonylation was significantly greater in OBs of 20- vs. 1.5-month-old mice. Aldolase 1 (ALDO1) showed significantly more carbonylation in OBs from 20- vs. 1.5-month-old mice; heat shock protein 9A and dihydropyrimidinase-like 2 showed significantly less. Several previously investigated proteins were also carbonylated, including ferritin heavy chain (FTH). Nitration, identified by 3-nitrotyrosine immunoreactivity, was least abundant at 1.5 months, intermediate at 6 months, and greatest at 20 months and was localized primarily in blood vessels. Proteins that were specific targets of oxidation were also localized: ALDO1 in astrocytes of the granule cell layer and FTH in mitral/tufted cells. These results indicate that specific carbonylated proteins, including those in astrocytes and mitral/tufted neurons, and nitrated proteins in the vasculature are molecular substrates of age-related olfactory dysfunction.

Identification of gene expression patterns using planned linear contrasts.

BACKGROUND: In gene networks, the timing of significant changes in the expression level of each gene may be the most critical information in time course expression profiles. With the same timing of the initial change, genes which share similar patterns of expression for any number of sampling intervals from the beginning should be considered co-expressed at certain level(s) in the gene networks. In addition, multiple testing problems are complicated in experiments with multi-level treatments when thousands of genes are involved. RESULTS: To address these issues, we first performed an ANOVA F test to identify significantly regulated genes. The Benjamini and Hochberg (BH) procedure of controlling false discovery rate (FDR) at 5% was applied to the P values of the F test. We then categorized the genes with a significant F test into 4 classes based on the timing of their initial responses by sequentially testing a complete set of orthogonal contrasts, the reverse Helmert series. For genes within each class, specific sequences of contrasts were performed to characterize their general 'fluctuation' shapes of expression along the subsequent sampling time points. To be consistent with the BH procedure, each contrast was examined using a stepwise Studentized Maximum Modulus test to control the gene based maximum family-wise error rate (MFWER) at the level of alphanew determined by the BH procedure. We demonstrated our method on the analysis of microarray data from murine olfactory sensory epithelia at five different time points after target ablation. CONCLUSION: In this manuscript, we used planned linear contrasts to analyze time-course microarray experiments. This analysis allowed us to characterize gene expression patterns based on the temporal order in the data, the timing of a gene's initial response, and the general shapes of gene expression patterns along the subsequent sampling time points. Our method is particularly suitable for analysis of microarray experiments in which it is often difficult to take sufficiently frequent measurements and/or the sampling intervals are non-uniform.

Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages.

Aged humans and rodents are susceptible to infection with Streptococcus pneumoniae bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines and increased production of interleukin (IL)-10 by macrophages (Mphi) from aged mice. To understand the molecular basis of cytokine dysregulation in aged mouse Mphi, a microarray analysis was performed on RNA from resting and lipopolysaccharide (LPS)-stimulated Mphi from aged and control mice using the Affymetrix Mouse Genome 430 2.0 gene chip. Two-way ANOVA analysis demonstrated that at an overall P < 0.01 level, 853 genes were regulated by LPS (169 in only the young, 184 in only the aged, and 500 in both). Expression analysis of systematic explorer revealed that immune response (proinflammatory chemokines, cytokines, and their receptors) and signal transduction genes were specifically reduced in aged mouse Mphi. Accordingly, expression of Il1 and Il6 was reduced, and Il10 was increased, confirming our previous results. There was also decreased expression of interferon-gamma. Genes in the Toll-like receptor-signaling pathway leading to nuclear factor-kappaB activation were also down-regulated but IL-1 receptor-associated kinase 3, a negative regulator of this pathway, was increased in aged mice. An increase in expression of the gene for p38 mitogen-activated protein kinase (MAPK) was observed with a corresponding increase in protein expression and enzyme activity confirmed by Western blotting. Low doses of a p38 MAPK inhibitor (SB203580) enhanced proinflammatory cytokine production by Mphi and reduced IL-10 levels, indicating that increased p38 MAPK activity has a role in cytokine dysregulation in the aged mouse Mphi.

Leptin regulates olfactory-mediated behavior in ob/ob mice.

We have investigated olfactory-mediated pre-ingestive behavior in leptin (ob/ob) and leptin receptor (db/db) mutant mice compared to age- and gender-matched wild-type (wt) mice. Olfactory-mediated behavior was tested using a buried food paradigm 5 times/day at 2-h intervals for 6 days. Mean food-finding times of ob/ob and db/db mice were approximately 10 times shorter than those of wt mice. To test the effect of leptin replacement in ob/ob mice, leptin (1 or 5 microg/g body weight in sterile saline) or carrier was injected i.p. once daily prior to testing. Mean food finding times in ob/ob mice injected with carrier or with 1 microg/g leptin were similar and were 2-3 times faster than in wt mice. Mean food finding times in ob/ob mice injected with 5 microg/g leptin tripled compared to carrier-injected ob/ob mice and were of the same order of magnitude as those of wt mice, suggesting functional leptin replacement. A 3-factor repeated measures ANOVA demonstrated significant differences between the 6 cohorts (P = 0.0001), food finding times (P< or = 0.0001), and cohort by day interaction (P< or = 0.0001). Post hoc tests suggested that the ob/ob+5 mug/g leptin cohort performed more like the wt cohort in the food-finding test than like the ob/ob or ob/ob+carrier cohort. Potential local sites of leptin production and action were identified with immunohistochemistry and in situ hybridization in epithelial and gland cells of the olfactory and nasal mucosae. Our results strongly suggest that leptin acting through leptin receptors modulates olfactory-mediated pre-ingestive behavior.

Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice.

The brain is susceptible to oxidative stress, which is associated with age-related brain dysfunction, because of its high content of peroxidizable unsaturated fatty acids, high oxygen consumption per unit weight, high content of key components for oxidative damage, and the relative scarcity of antioxidant defense systems. Protein oxidation, which results in functional disruption, is not random but appears to be associated with increased oxidation in specific proteins. By using a proteomics approach, we have compared the protein levels and specific protein carbonyl levels, an index of oxidative damage in the brains of old mice, to these parameters in the brains of young mice and have identified specific proteins that are altered as a function of aging. We show here that the expression levels of dihydropyrimidinase-like 2 (DRP2), alpha-enolase (ENO1), dynamin-1 (DNM1), and lactate dehydrogenase 2 (LDH2) were significantly increased in the brains of old versus young mice; the expression levels of three unidentified proteins were significantly decreased. The specific carbonyl levels of beta-actin (ACTB), glutamine synthase (GS), and neurofilament 66 (NF-66) as well as a novel protein were significantly increased, indicating protein oxidation, in the brains of old versus young mice. These results were validated by immunochemistry. In addition, enzyme activity assays demonstrated that oxidation was associated with decreased GS activity, while the activity of lactate dehydrogenase was unchanged in spite of an up-regulation of LDH2 levels. Several of the up-regulated and oxidized proteins in the brains of old mice identified in this report are known to be oxidized in neurodegenerative diseases as well, suggesting that these proteins may be particularly susceptible to processes associated with neurodegeneration. Our results establish an initial basis for understanding protein alterations that may lead to age-related cellular dysfunction in the brain.

Proteomic identification of differentially expressed proteins in the aging murine olfactory system and transcriptional analysis of the associated genes.

Decline in olfactory ability has been associated with aging as well as neurodegenerative disorders. The aim of this study was to gain fundamental insight into molecular events associated with the aging olfactory system. We report a comparative proteomic analysis of the olfactory epithelium (OE) and olfactory bulb (OB) of old (80-week old) and young (6-week old) mice with further analysis of age-related differences in differentially expressed proteins at the mRNA level using real-time RT-PCR. Nine proteins in the OE and 20 in the OB were differentially expressed in old and young mice; of these, aldolase 1, peptidyl prolyl isomerase A, mitochondrial aconitase 2, mitochondrial aldehyde dehydrogenase 2 and albumin 1 were identified in the OE; and ATP synthase isoform 1, enolase 1, ferritin heavy chain, malate dehydrogenase 1, tropomyosin alpha 3 chain and dynamin 1 were identified in the OB. At the transcriptional level, aconitase 2 in the OE and ferritin heavy chain 1 in the OB were differentially expressed with aging, in concordance with the proteomic data. Our results demonstrate an altered proteomic profile of the aged murine olfactory system. The identified proteins fall into three broadly defined functional categories: (i) metabolism, (ii) transport/motility and (iii) stress response. Our transcriptional analysis provides insight into possible mechanisms by which protein expression may be regulated in the OE and OB. The results are discussed in relation to the decrement in olfactory sensitivity with aging.

Quadratic regression analysis for gene discovery and pattern recognition for non-cyclic short time-course microarray experiments.

BACKGROUND: Cluster analyses are used to analyze microarray time-course data for gene discovery and pattern recognition. However, in general, these methods do not take advantage of the fact that time is a continuous variable, and existing clustering methods often group biologically unrelated genes together. RESULTS: We propose a quadratic regression method for identification of differentially expressed genes and classification of genes based on their temporal expression profiles for non-cyclic short time-course microarray data. This method treats time as a continuous variable, therefore preserves actual time information. We applied this method to a microarray time-course study of gene expression at short time intervals following deafferentation of olfactory receptor neurons. Nine regression patterns have been identified and shown to fit gene expression profiles better than k-means clusters. EASE analysis identified over-represented functional groups in each regression pattern and each k-means cluster, which further demonstrated that the regression method provided more biologically meaningful classifications of gene expression profiles than the k-means clustering method. Comparison with Peddada et al.'s order-restricted inference method showed that our method provides a different perspective on the temporal gene profiles. Reliability study indicates that regression patterns have the highest reliabilities. CONCLUSION: Our results demonstrate that the proposed quadratic regression method improves gene discovery and pattern recognition for non-cyclic short time-course microarray data. With a freely accessible Excel macro, investigators can readily apply this method to their microarray data.

Temporal profiling of gene expression during neurogenesis and remodeling in the olfactory epithelium at short intervals after target ablation.

Neurogenesis in the olfactory epithelium (OE) is induced by olfactory bulbectomy (OBX), which effectively axotomizes olfactory sensory neurons (OSNs) and removes their synaptic targets, resulting in apoptosis. We used Affymetrix high-density oligonucleotide arrays to investigate changes in gene expression during initiation of signaling in pathways that regulate apoptosis and neurogenesis in the murine OE at 2, 8, 16, and 48 hr after bilateral OBX compared to that in sham-operated controls. We focused on regulation of a defined set of genes associated with apoptosis, stem/progenitor cell regulation, and cell cycle progression because of the activation of these processes in OE degeneration and remodeling after OBX. After data scrubbing and categorical analysis, one-way analysis of variance identified 72 genes (4.9% of the present known genes) as being regulated significantly (P < 0.05) at one or more points; 50 were defined as regulated differentially with the false discovery rate at 10%. Significant changes in gene expression occurred in all categories as early as 2 hr post-OBX, with the greatest number of differentially regulated genes at 16 and 48 hr. Hierarchical cluster analysis and correlation coefficients were used to identify similarities in patterns of gene expression changes within and across categories. Validation was carried out with SuperArray macroarrays and real-time RT-PCR. Our results confirmed the participation of many genes in known signaling pathways and identified changes in the expression of 42 genes not identified previously as participating in apoptosis and neurogenesis in the OE. Additionally, our analyses indicated the early involvement of genes regulating cytoskeletal reorganization and angiogenesis in the response to OBX. These studies are an important first step in defining early time-dependent changes in gene expression after target ablation that lead to neurogenesis in the olfactory sensory epithelium.

Target ablation-induced regulation of macrophage recruitment into the olfactory epithelium of Mip-1alpha-/- mice and restoration of function by exogenous MIP-1alpha.

The chemokine macrophage inflammatory protein (MIP)-1alpha recruits macrophages to sites of epithelial remodeling. We showed previously that mRNA and protein levels of MIP-1alpha in the olfactory epithelium (OE) increased significantly at 3 days after bilateral olfactory bulbectomy (OBX). The first aim of this study was to investigate the effect of the absence of MIP-1alpha on macrophage recruitment to the OE 3 days after OBX in Mip-1alpha(-/-) mice compared with C57BL/6 mice and to test whether chemokine function could be restored by MIP-1alpha protein injection into Mip-1alpha(-/-) mice. OBX was performed on C57BL/6 and Mip-1alpha(-/-) mice. The mice received six subcutaneous injections at 12-h intervals of either 10 mug/ml MIP-1alpha protein in carrier or carrier only. Macrophage recruitment was evaluated with antibodies to CD68 for all macrophages and F4/80 for activated macrophages. Compared with C57BL/6 mice, at 3 days post-OBX the numbers of CD68(+) and F4/80(+) macrophages were significantly lower in carrier-injected Mip-1alpha(-/-) mice and were comparable in MIP-1alpha protein-injected Mip-1alpha(-/-) mice. The second aim was to determine the identity of genes regulated at 3 days post-OBX in the OE of carrier-injected Mip-1alpha(-/-) mice compared with carrier-injected C57BL/6 mice. Total RNA from the OE was hybridized to Affymetrix microarrays. A number of chemokine-, cytokine-, and growth factor-related genes were significantly regulated in the Mip-1alpha(-/-) mice and were restored in MIP-1alpha protein-injected Mip-1alpha(-/-) mice. The results illustrated that MIP-1alpha played a key role in recruitment of macrophages to the OE and provided insight into the genomic regulation involved in OE remodeling.

In silico analysis of gene expression profiles in the olfactory mucosae of aging senescence-accelerated mice.

We utilized high-density Affymetrix oligonucleotide arrays to investigate gene expression in the olfactory mucosae of near age-matched aging senescence-accelerated mice (SAM). The senescence-prone (SAMP) strain has a significantly shorter lifespan than does the senescence-resistant (SAMR) strain. To analyze our data, we applied biostatistical methods that included a correlation analysis to evaluate sources of methodologic and biological variability; a two-sided t-test to identify a subpopulation of Present genes with a biologically relevant P-value <0.05; and a false discovery rate (FDR) analysis adjusted to a stringent 5% level that yielded 127 genes with a P-value of <0.001 that were differentially regulated in near age-matched SAMPs (SAMP-Os; 13.75 months) compared to SAMRs (SAMR-Os, 12.5 months). Volcano plots related the variability in the mean hybridization signals as determined by the two-sided t-test to fold changes in gene expression. The genes were categorized into the six functional groups used previously in gene profiling experiments to identify candidate genes that may be relevant for senescence at the genomic and cellular levels in the aging mouse brain (Lee et al. [2000] Nat Genet 25:294-297) and in the olfactory mucosa (Getchell et al. [2003] Ageing Res Rev 2:211-243), which serves several functions that include chemosensory detection, immune barrier function, xenobiotic metabolism, and neurogenesis. Because SAMR-Os and SAMP-Os have substantially different median lifespans, we related the rate constant alpha in the Gompertz equation on aging to intrinsic as opposed to environmental mechanisms of senescence based on our analysis of genes modulated during aging in the olfactory mucosa.

Analysis of oligonucleotide array experiments with repeated measures using mixed models.

BACKGROUND: Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease) or absence (Control) of the disease, and brain regions including olfactory bulb (OB) or cerebellum (CER). In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. RESULTS: In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH) procedure of controlling false discovery rate (FDR) at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the alpha-level (alphanew = 0.0033) determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD) procedure at the level of alphanew to control the family-wise error rate (FWER) for each gene examined. CONCLUSIONS: A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

Age-related trends in gene expression in the chemosensory-nasal mucosae of senescence-accelerated mice.

We have utilized high-density GeneChip oligonucleotide arrays to investigate the use of the senescence-accelerated mouse (SAM) as a biogerontological resource to identify patterns of gene expression in the chemosensory-nasal mucosa. Gene profiling in chronologically young and old mice of the senescence-resistant (SAMR) and senescence-prone (SAMP) strains revealed 133 known genes that were modulated by a three-fold or greater change either in one strain or the other or in both strains during aging. We also identified known genes in our study which based on their encoded proteins were identified as aging-related genes in the aging neocortex and cerebellum of mice as reported by Lee et al. (2000) [Nat. Genet. 25 (2000) 294]. Changes in gene profiles for chemosensory-related genes including olfactory and vomeronasal receptors, sensory transduction-associated proteins, and odor and pheromone transport molecules in the young SAMR and SAMP were compared with age-matched C57BL/6J mice. An analysis of known gene expression profiles suggests that changes in the expression of immune factor genes and genes associated with cell cycle progression and cell death were particularly prominent in the old SAM strains. A preliminary cellular validation study supported the dysregulation of cell cycle-related genes in the old SAM strains. The results of our initial study indicated that the use of the SAM models of aging could provide substantive information leading to a more fundamental understanding of the aging process in the chemosensory-nasal mucosa at the genomic, molecular, and cellular levels.

Chemokine regulation of macrophage recruitment into the olfactory epithelium following target ablation: involvement of macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1.

Target ablation by olfactory bulbectomy synchronizes the degenerative cell death of olfactory receptor neurons (ORNs), infiltration of macrophages, and proliferation of progenitor cells, leading to neurogenesis, ORN replacement, and regeneration of the sensory epithelium. Although macrophages participate in the degenerative and regenerative events, little is known of the molecular and cellular mechanisms associated with their recruitment during the earliest period following target ablation. Macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1), which are members of the CC or beta-chemokine subfamily, are chemoattractants for monocytes/macrophages. Shortly after target ablation, the protein and mRNA levels for MIP-1alpha and MCP-1 were up-regulated, showing peak expression levels from 16 hr to 3 days post-OBX; this coincided with the pattern of infiltration of activated F4/80(+) macrophages. The mRNAs for MIP-1alpha and MCP-1, as well as their cognate receptors CCR1 and CCR2, respectively, were localized in resident and infiltrating macrophages in numbers commensurate with those of F4/80-immunopositive macrophages in adjacent tissue sections. The mRNA(+) macrophages were localized within olfactory epithelial compartments that corresponded with their proposed functions associated with phagocytosis, proliferation, and infiltration. Our data support the hypothesis that MIP-1alpha and MCP-1 are chemoattractant chemokines associated with the recruitment of macrophages into the olfactory epithelium shortly after target ablation.

Expression of TGF-beta type II receptors in the olfactory epithelium and their regulation in TGF-alpha transgenic mice.

Numerous in vitro studies of neurogenesis of olfactory receptor neurons (ORNs) suggest that transforming growth factor (TGF)-beta promotes the maturation/differentiation of olfactory progenitors. We demonstrate that in vivo both mature and immature ORNs, and possibly a basal neuronal progenitor cell, express the TGF-beta type II receptor (TGF-betaRII), suggesting that these cells are targets for TGF-beta signaling. In a previous study of neurogenesis in the OE of TGF-alpha overexpressing transgenic (T) mice, we observed an apparent reduction in the expression of olfactory marker protein (OMP), a marker of terminal differentiation in ORNs in T mice compared to nontransgenic (NT) littermate controls; this was confirmed by Western blotting and immunohistochemistry. In contrast, there was no apparent difference between T and NT mice in the intensity of immunoreactivity for a neuronal marker, protein gene product 9.5. Because TGF-alpha overexpression has been reported to affect TGF-beta signaling in other epithelia, we compared the expression of the TGF-beta type II receptor (TGF-betaRII) in T and NT mice. The intensity of TGF-betaRII immunoreactivity on ORNs was substantially reduced in T compared to NT mice. Similar reductions in TGF-betaRII expression in vomeronasal receptor neurons and in other epithelia in the nasal cavity of T mice were also observed. Taken together, these results indicate that TGF-beta signaling regulates terminal differentiation of ORNs in vivo and suggest ways in which interactions between TGF-alpha and TGF-beta signaling pathways may interact in the OE.

Olfactory bulbectomy protects hippocampal pyramidal neurons against excitotoxic death.

The olfactory system is functionally linked to the hippocampus, and odors can modify the activity of hippocampal neurons. Because hippocampal neurons are selectively vulnerable to death in several prominent neurodegenerative conditions, we tested the hypothesis that activity in olfactory pathways can modify the sensitivity of hippocampal neurons to excitotoxic damage. We report that rats subjected to olfactory bulbectomy exhibit a decrease in the vulnerability of hippocampal pyramidal neurons to excitotoxic injury. Four-month-old male Sprague-Dawley rats were subjected to bilateral olfactory bulbectomy or a sham operation. Three months later the rats were given a unilateral infusion of kainic acid in the dorsal hippocampus and were euthanized 24 h later. There was a threefold increase in the number of CA3 neurons that survived kainic acid administration in the bulbectomized rats compared to sham-operated rats. These findings provide the first evidence that olfactory input affects the vulnerability of neurons to excitotoxic death.

Leukemia inhibitory factor mRNA expression is upregulated in macrophages and olfactory receptor neurons after target ablation.

After target ablation by olfactory bulbectomy (OBX), the murine olfactory epithelium (OE) undergoes degenerative changes leading to apoptosis of olfactory receptor neurons (ORNs) followed by regenerative changes that include proliferation of progenitor cells leading to neurogenesis and ORN replacement. Macrophages recruited to the OE after OBX are involved in both the degenerative and regenerative processes. Relative quantitative RT-PCR was used to demonstrate that within hours of OBX, mRNAs encoding three key components in the leukemia inhibitory factor (LIF) signaling pathway, including LIF, LIF receptor (LIFR), and STAT3, as well as cyclin D1, a growth factor sensor indicative of progenitor cell transformation, were upregulated. These mRNAs reached peak levels of expression on or before day 3 post-OBX, coincident with the peak time for macrophage recruitment and progenitor cell proliferation. Cells expressing LIF mRNA in the OE of mice at 3 days post-OBX, the time point at which LIF mRNA expression peaked, were identified using non-isotopic in situ hybridization. LIF mRNA was localized in infiltrating macrophages; near-adjacent sections exhibited macrophages immunoreactive for F4/80, a marker for activated macrophages, in numbers commensurate with those expressing LIF mRNA. LIF mRNA was also localized in surviving ORNs, identified by their expression of olfactory marker protein (OMP) mRNA and protein in near-adjacent sections. Our data suggest that LIF functions as a mitogen originating from recruited macrophages through an intercellular signaling pathway that stimulates proliferation of progenitor cells leading to neurogenesis and regeneration, and as an intracellular survival factor for traumatized ORNs.