DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition, patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD, we determined whether inefficient BER due to reduced DNA polymerase‐β (Polβ) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild‐type and 3xTgAD mice with normal or reduced Polβ levels. Compared to wild‐type control mice, Polβ heterozygous (Polβ^+/?), and 3xTgAD mice, 3xTgAD/Polβ^+/? mice exhibited impaired performance in a buried food test of olfaction. Polβ deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However, numbers of newly generated neurons were reduced by approximately 25% in Polβ^+/? and 3xTgAD mice, and by over 60% in the 3xTgAD/Polβ^+/? mice compared to wild‐type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polβ^+/? mice compared to 3xTgAD mice. Levels of amyloid β‐peptide (Aβ) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polβ^+/? mice, and cultured Polβ‐deficient neurons exhibited increased vulnerability to Aβ‐induced death. Olfactory deficit is an early sign in human AD, but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons, and suggest a mechanism underlying early olfactory impairment in AD.     

刺猬嗅球冬眠期与非冬眠期c-Fos 表达的差异

利用免疫组化法检测c-Fos 蛋白在不同季节刺猬嗅球各层次的表达差异,探讨c-Fos、嗅觉、冬眠三者的关系。分别选取春、夏、秋、冬四个季节各6 只野生健康刺猬,固定剥离嗅球,石蜡切片,免疫组化显色,拍片,载入Motic Images Advanced 3.2 软件,测量四个季节刺猬嗅球各层次c-Fos 的表达率,将结果载入GraphPadPrism4 软件分析,Microsoft Excel 作图。结果表明:c-Fos 蛋白在成年刺猬嗅球各层均有不同程度的表达,阴性对照不着色,且表现出明显的季节性差异。1)与秋季相比,冬眠期c-Fos 蛋白在刺猬嗅球各层次的表达均有极显著的降低(P <0.01);2)与夏季相比,冬眠期c-Fos 蛋白在外网丛层、僧帽细胞层、颗粒细胞层的表达有极显著降低(P < 0. 01),在嗅神经层、嗅小球层、室管膜层的表达也有显著降低(P < 0. 05);3)与冬眠期相比,春季c-Fos 蛋白在嗅小球层、僧帽细胞层、颗粒细胞层的表达有极显著的升高(P <0. 01),在嗅神经层、外网丛层、室管膜层的表达却没有显著变化(P ﹥ 0.05);4)嗅神经层c-Fos 的表达在春季显著低于秋季,夏季与秋季没有显著差异。颗粒细胞层夏季显著低于秋季(P < 0.05)。秋季c-Fos 在其余各层次的表达与春季、夏季相比都有极显著的提高(P <0.01)。结论:秋季刺猬嗅球神经元最活跃,嗅觉最灵敏,冬眠期刺猬嗅球活跃性大大降低,嗅觉系统最迟钝。c-Fos 在刺猬嗅球中的强表达表明其在嗅觉信息的传递中可能发挥一定作用,c-Fos 表达率的显著季节性差异揭示了刺猬嗅球的活跃性与其冬眠具有一定的相关性。     

Role of olfaction in food preference as evaluated in an animal model.

Food preference in individual animals is regulated by brain activity. Two murine model systems for investigating food preference were developed by focusing on fruit juices. In a home-cage, two-bottle test, the volume of apple juice consumed was found to be much larger than that of orange juice. In a two-nozzle "Drinkometer" test, by which each mouse was kept in a 38 cm (W) x 32 cm (D) cage and each drinking event was recorded by an electronic "Drinkometer" device, it was again found that the mice preferred drinking apple juice to orange juice. To elucidate the role of olfaction in this food preference, mice were subjected to an olfactory bulbectomy to remove the olfaction capability. In the home-cage two-bottle test, the preference for apple juice over orange juice was apparent even after the olfactory bulbectomy, indicating that olfaction was not essential for the formation of food preference behavior. In contrast, in the two-nozzle "Drinkometer" test, the preference for apple juice over orange juice was found to be abrogated by this surgery, implying the involvement of olfaction-based memory on food preference behavior.     

Stam2 expression pattern during embryo development

STAM2 is a tyrosine-phosphorylated protein suggested to be involved in cargo selection during endocytic pathway, regulation of exocytosis and intracellular signaling. Gene trap method was used to create via insertional mutagenesis a mutant mouse line with integration of promoterless βgeo (lacZ-neomycin phosphotransferase fusion) gene in the second intron of Stam2 gene, enabling analysis of its in vivo expression and function. The inserted β-galactosidase (lacZ) reporter gene was used to reveal Stam2 expression during development. Stam2 in situ RNA hybridization and immunostaining confirmed the observed β-galactosidase activity reflecting high Stam2 expression. The homozygous mutant mice showed no overt phenotypic alterations. Stam2 expression was detected after E9.5 in the gut, notochord, neural tube and heart. In the nervous system it was located in the floor, roof and basal plates of the developing neural tube, and in the developing cortex, hippocampus and olfactory bulbs. Toward the end of gestation, Stam2 expression appeared in the testis and ovary, lungs, nasal cavity epithelium, kidneys, urogenital sinus, intestine, pancreas, pituitary and adrenal glands, muscles, brown adipose tissue, skin and epithelium of the tongue and oral cavity.     

Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice

Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.     

Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions

We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss.     

Use of ultrasonic vocalizations to assess olfactory detection in mouse pups treated with 3-methylindole

Altricial mammals use olfaction long before the olfactory bulb has reached its anatomically mature state. Indeed, while audition and vision are still not functional, the olfactory system of newborn animals can clearly process distinct odorant molecules. Although several previous studies have emphasized the important role that olfaction plays in early critical functions, it has been difficult to develop a sensitive and reliable test to precisely quantify olfactory ability in pups. One difficulty in determining early sensory capabilities is the rather limited behavioral repertory of neonates. The present study examines the use of ultrasonic vocalizations emitted by isolated rodent pups as a potential index of odor detection in newborn mice. As early as postnatal day 2, mice reliably decrease their emission of ultrasonic calls in response to odor exposure to the bedding of adult male mice but not in response to clean bedding odors or to non-social odorant molecules. A toxin known to damage the olfactory epithelium in adult, the 3-methylindole, impairs the ultrasonic call responses triggered by exposure to male bedding, thus confirming the efficiency of this olfactotoxin on mice pups. The administration of 3-methylindole severely reduced the life expectancy of the majority of subjects. This result is discussed according to the critical role of olfaction in nipple-seeking behavior in mouse pups.     

In Utero and Lactational Lanthanum Exposure Induces Olfactory Dysfunction Associated with Downregulation of βIII-tubulin and Olfactory Marker Protein in Young Rats

We evaluated the role of βIII-tubulin in the morphology of olfactory receptor neuron (ORN) and olfactory dysfunction in offspring caused by prenatal and postnatal lanthanum exposure. Pregnant rats were exposed to 0.25% lanthanum chloride in drinking water from gestational day (GD) 7 until postnatal day 21. From postnatal day 23 until postnatal day 28, pups were examined with buried food pellet and olfactory maze test. The ultrastructural features of ORNs in the olfactory epithelium (OE) were observed by transmission electron microscope. The expression of βIII-tubulin and olfactory marker protein (OMP) in the tissue sections and homogenates of OE were, respectively, measured by immunodetection and western blot. Behavioral analysis of olfaction showed that lanthanum chloride exposure induced olfactory dysfunction. Offsprings exposed to lanthanum chloride showed enlarged ORN knobs and a decreased number of cilia. In addition, the levels of OMP and βIII-tubulin expression in lanthanum chloride exposure offsprings significantly decreased. Developmental lanthanum exposure could impair olfaction, and this deficit may be attributed to the downregulation of βIII-tubulin and OMP in the OE.     

Impaired Sense of Smell in a Drosophila Parkinson’s Model

Parkinson’s disease (PD) is one of the most common neurodegenerative disease characterized by the clinical triad: tremor, akinesia and rigidity. Several studies have suggested that PD patients show disturbances in olfaction at the earliest onset of the disease. The fruit fly Drosophila melanogaster is becoming a powerful model organism to study neurodegenerative diseases. We sought to use this system to explore olfactory dysfunction, if any, in PINK1 mutants, which is a model for PD. PINK1 mutants display many important diagnostic symptoms of the disease such as akinetic motor behavior. In the present study, we describe for the first time, to the best of our knowledge, neurophysiological and neuroanatomical results concerning the olfactory function in PINK1 mutant flies. Electroantennograms were recorded in response to synthetic and natural volatiles (essential oils) from groups of PINK1 mutant adults at three different time points in their life cycle: one from 3–5 day-old flies, from 15–20 and from 27–30 days. The results obtained were compared with the same age-groups of wild type flies. We found that mutant adults showed a decrease in the olfactory response to 1-hexanol, α-pinene and essential oil volatiles. This olfactory response in mutant adults decreased even more as the flies aged. Immunohistological analysis of the antennal lobes in these mutants revealed structural abnormalities, especially in the expression of Bruchpilot protein, a marker for synaptic active zones. The combination of electrophysiological and morphological results suggests that the altered synaptic organization may be due to a neurodegenerative process. Our results indicate that this model can be used as a tool for understanding PD pathogensis and pathophysiology. These results help to explore the potential of using olfaction as a means of monitoring PD progression and developing new treatments.     

Cholecystokinin levels in prohormone convertase 2 knock-out mouse brain regions reveal a complex phenotype of region-specific alterations

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.     

Y1 receptors are critical for the proliferation of adult mouse precursor cells in the olfactory neuroepithelium

While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.     

Effects of zinc gluconate and 2 other divalent cationic compounds on olfactory function in mice

Intranasal application of zinc gluconate has commonly been used to treat the common cold. The safety of this treatment, however, has come into question recently. In addition to a United States recall of a homeopathic product that contains zinc gluconate, abundant literature reports cytotoxic effects of zinc on the olfactory epithelium. Additional research suggests that divalent cations (such as zinc) can block ion channels that facilitate the transduction of odors into electrical signals on the olfactory epithelium. The purpose of the current study was 2-fold: to confirm whether zinc gluconate causes anosmia and to reveal whether any other divalent cationic compounds produce a similar effect. Groups of mice underwent a buried food-pellet test to gauge olfactory function and then were nasally irrigated with 1 of 3 divalent cationic compounds. When tested after treatment, mice irrigated with zinc gluconate and copper gluconate experienced a marked increase in food-finding time, indicating that they had lost their ability to smell a hidden food source. Control mice irrigated with saline had a significantly lower increase in times. These results confirm that zinc gluconate can cause anosmia and reveal that multiple divalent cations can negatively affect olfaction.     

Preference for corn oil in olfactory-blocked mice in the conditioned place preference test and the two-bottle choice test

We studied the effects of olfactory stimuli on preference for corn oil in mice. In the conditioned place preference test, voluntary intake of 100% corn oil by both olfactory normal and ZnSO4-induced olfactory-blocked (anosmic) mice resulted in their place preference for the corn oil-related box. In the olfactory normal mice, place preference was also observed by voluntary intake of linoleic acid as well as of corn oil. In the two-bottle choice test, normal mice showed significant preference for test fluids that contained corn oil at all concentrations (1-10%) tested relative to vehicle alone. However, the lower concentrations (1 and 3%) of corn oil were not preferred in the anosmic mice. These results suggested that stimuli other than olfaction contributed to the rewarding effects of corn oil, but at lower concentrations olfactory stimuli might act as a signal for the oil.     

Excessive microglial activation aggravates olfactory dysfunction by impeding the survival of newborn neurons in the olfactory bulb of Niemann–Pick disease type C1 mice

Progressive olfactory impairment is one of the earliest markers of neurodegeneration. However, the underlying mechanism for this dysfunction remains unclear. The present study investigated the possible role of microgliosis in olfactory deficits using a mouse model of Niemann–Pick disease type C1 (NPC1), which is an incurable neurodegenerative disorder with disrupted lipid trafficking. At 7 weeks of age, NPC1 mutants showed a distinct olfactory impairment in an olfactory test compared with age-matched wild-type controls (WT). The marked loss of olfactory sensory neurons within the NPC1 affected olfactory bulb (NPC1-OB) suggests that NPC1 dysfunction impairs olfactory structure. Furthermore, the pool of neuroblasts in the OB was diminished in NPC1 mice despite the intact proliferative capacity of neural stem/progenitor cells in the subventricular zone. Instead, pro-inflammatory proliferating microglia accumulated extensively in the NPC1-OB as the disease progressed. To evaluate the impact of abnormal microglial activation on olfaction in NPC1 mice, a microglial inhibition study was performed using the anti-inflammatory agent Cyclosporin A (CsA). Importantly, long-term CsA treatment in NPC1 mice reduced reactive microgliosis, restored the survival of newly generated neurons in the OB and improved overall performance on the olfactory test. Therefore, our study highlights the possible role of microglia in the regulation of neuronal turnover in the OB and provides insight into the possible therapeutic applications of microglial inhibition in the attenuation or reversal of olfactory impairment.     

Olfactory conditioning experiments in a food-searching passerine bird in semi-natural conditions

Because passerine birds have a very small relative olfactory bulb size, they have been considered to have weak olfactory capacities for decades. Recent investigations however suggest that breeding female blue tits (Parus caeruleus) are sensitive to lavender odour in the reproductive context of building and maintaining the nest. Here, we present results of an olfactory conditioning experiment in blue tits held in semi-natural conditions during the breeding season. We show that captive male blue tits, trained to associate lavender odour with a food reward, are more attracted to an empty feeder box emitting lavender odour than an odourless empty feeder box. Females did not distinguish significantly between empty feeders with and without lavender odour during the test phase, although they responded positively at the end of the training phase. These results suggest that male blue tits can use olfaction in a context not related to nest building. Additional experiments will be required to better understand the observed sex differences in response to the experimental set up, and in what context free-ranging individuals use olfaction.     

p59fyn and pp60 c-src modulate axonal guidance in the developing mouse olfactory pathway

The Src-family tyrosine kinases p59^fyn and pp60^c-src are localized on axons of the mouse olfactory nerve during the initial stages of axonal growth, but their functional roles remain to be defined. To study the role of these kinases, we analyzed the trajectory of the olfactory nerve in E11.5 homozygous null mutant mice lacking single src or fyn genes and double mutants lacking both genes. Primary olfactory axons of single and double mutants exited the olfactory epithelium and projected toward the telencephalon, but displayed differences in fasciculation. The fyn-minus olfactory nerve had significantly more fascicles than the src-minus nerve. Most strikingly, the primary olfactory nerve of src/fyn double mutants showed the greatest degree of defasciculation. These defects, identified by NCAM labeling, were not due to apparent changes in the size of the olfactory epithelium. With the exception of the src-minus mice, which had fewer fascicles than the wild type, no obvious differences were observed in coalescence of vomeronasal axons from mutant mice. The mesenchyme of the double and single mutants exhibited only subtle changes in laminin and fibronectin staining, indicating that the adhesive environment of the mesenchyme may contribute in part to defects in fasciculation. The results suggest that signaling pathways mediated by p59^fyn and pp60^c-src contribute to the appropriate fasciculation of axons in the nascent olfactory system, and comprise partially compensatory mechanisms for axonal adhesion and guidance. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 53–63, 1998     

Dab1 (Disable homolog-1) reelin adaptor protein is overexpressed in the olfactory bulb at early postnatal stages

Dab1 mediates reelin signalling and plays critical roles in early brain development such as the stereotypical positioning of neurons in the brain. The olfactory bulb undergoes a prominent layering reorganization, but shows not apparent differences between wild type and reeler in the layer organization. Therefore, an accurate regional and cellular simultaneous analysis of these molecules becomes essential to clarify the role played by Dab1 upon Reelin effect. The present study reveals a strong and consistent Dab1 mRNA and protein expressions, throughout the olfactory bulb layers in both wild type and reeler mice. In addition, noteworthy is the pattern of Dab1 location within cell nuclei in both strains. Furthermore, a temporal increment of Dab1 expression levels is detected from P0 to P15 in both strains, being the protein quantity higher in reeler than in wild type mice. Altogether, our results revealed that Reln acts directly from projection neurons via the production of different Reln fragments. Changes in the pattern of Dab1 expression could reflect an alternative Reln function in postnatal and adult stages, besides a possible regulation of Dab1 by other molecules distinct to Reln.     

Diademed sifakas (Propithecus diadema) use olfaction to forage for the inflorescences of subterranean parasitic plants (Balanophoraceae: Langsdorffia sp., and Cytinaceae: Cytinus sp.)

Primates usually locate food resources using visual cues and memory, yet the potential for olfactory-guided (or olfactory-assisted) food location remains relatively unexplored. Here we report observations of wild Propithecus diadema that strongly suggest that olfaction is used to locate the inflorescences of two subterranean parasitic plant species (Langsdorffia sp. and Cytinus sp.). These valued but seasonal food resources are found obscured in leaf litter, and sifakas spend considerable time on the ground engaged in what appears to be olfactory exploration before they locate the inflorescences. Because they are visually obscured and occur within a substrate that is rarely used by sifakas, accidental discovery of these resources seems unlikely. Individuals may learn to exploit them by watching conspecifics.     

Mouse cyp2g1 gene: promoter structure and tissue-specific expression of a cyp2g1-lacz fusion gene in transgenic mice.

The structure of the mouse Cyp2g1 gene was determined to identify regulatory regions important for its olfactory mucosa-specific expression. Two Cyp2g1 genomic clones were isolated and characterized. A 3.6-kilobase 5'-flanking sequence was used to prepare a Cyp2g1--LacZ fusion gene for transgenic mice production. Transgene expression, as determined by beta-galactosidase activity in tissue extracts, was detected in the olfactory mucosa, but not in any other tissues examined, in five different transgenic lines. Thus, the 3.6-kilobase fragment contained regulatory elements sufficient for olfactory mucosa-specific and proper developmental expression of the reporter gene. However, histological and immunohistochemical studies indicated that the expression of the transgene in the olfactory mucosa was patchy and the cellular expression patterns of the transgene did not exactly match that of the endogenous gene. These results implicate the presence of additional regulatory sequences that are necessary for the correct cell type-selectivity within the olfactory mucosa.