Expression of Myostatin in Neural Cells of the Olfactory System

Recent studies show that myostatin mRNA expression is found in some regions of the brain. However, the functional significance of this is currently unknown. We therefore investigated myostatin expression and function in the brain. In this study, we used immunohistochemistry, in situ hybridization, and RT-PCR analysis to reveal that myostatin is expressed in the mitral cells in the olfactory bulb (OB) and in neurons in the olfactory cortex (OC). Using 3D reconstruction, mitral cells positive for myostatin were positioned in the lateral and ventral regions of the OB. In contrast, myostatin-positive mitral cells were detected in mice at 2 weeks of age, but not on days 0 and 7 after birth. Activin receptor IIB, a myostatin receptor, was expressed in the OB, OC, hippocampus, and paraventricular thalamic nucleus. Moreover, c-Fos immunostaining in granule cells in the OB was augmented after intracerebroventricular injection of myostatin. These findings suggest that myostatin is localized in specific cells associated with the olfactory system of the brain and may act as a key inhibitor in cell and/or signal development of the olfactory system.     

BDNF promoter-mediated beta-galactosidase expression in the olfactory epithelium and bulb

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the generation and differentiation of new olfactory sensory neurons (OSNs) and in the regulation of branching of OSN axons in their target glomeruli. However, previous reports of BDNF mRNA and protein expression in olfactory epithelium and olfactory bulb (OB) have been inconsistent, raising questions on the proposed roles for BDNF. Here, we report on beta-galactosidase (beta-gal) expression in adult gene-targeted mice where the BDNF promoter drives expression of the Escherichia coli lacZ gene (BDNF(lacZneo) mice). We find that beta-gal is expressed in a small subset of OSNs with axons that reach the olfactory nerve layers throughout the OB. In the OB, we find expression of beta-gal in gamma-aminobutyric acidergic but not dopaminergic periglomerular cells and external tufted cells and in interneurons located in the mitral cell layer. Our results are inconsistent with the regulation of generation and differentiation of new OSNs elicited by the release of BDNF from horizontal basal cells. The results are consistent with a role for BDNF in competitive branching of OSN axons within the glomeruli of the OB.     

The localization of neuronal nitric oxide synthase may influence its role in neuronal precursor proliferation and synaptic maintenance

Neuronal nitric oxide synthase (nNOS) is implicated in some developmental processes, including neuronal survival, differentiation, and precursor proliferation. To define the roles of nNOS in neuronal development, we utilized the olfactory system as a model. We hypothesized that the role of nNOS may be influenced by its localization. nNOS expression was developmentally regulated in the olfactory system. During early postnatal development, nNOS was expressed in developing neurons in the olfactory epithelium (OE), while in the adult its expression was restricted to periglomerular (PG) cells in the olfactory bulb (OB). At postnatal week 1 (P1W), loss of nNOS due to targeted gene deletion resulted in a decrease in immature neurons in the OE due to decreased proliferation of neuronal precursors. While the pool of neuronal precursors and neurogenesis normalized in the nNOS null mouse by P6W, there was an overgrowth of mitral or tufted cells dendrites and a decreased number of active synapses in the OB. Cyclic GMP (cGMP) immunostaining was reduced in the OE and in the glomeruli of the OB at early postnatal and adult ages, respectively. Our results suggest that nNOS appears necessary for neurogenesis in the OE during early postnatal development and for glomerular organization in the OB in the adult. Thus, the location of nNOS, either within cell bodies or perisynaptically, may influence its developmental role.     

Patterns of heterogeneous expression of pannexin 1 and pannexin 2 transcripts in the olfactory epithelium and olfactory bulb

Pannexins form membrane channels that release biological signals to communicate with neighboring cells. Here, we report expression patterns of pannexin 1 (Panx1) and pannexin 2 (Panx2) in the olfactory epithelium and olfactory bulb of adult mice. In situ hybridization revealed that mRNAs for Panx1 and Panx2 were both expressed in the olfactory epithelium and olfactory bulb. Expression of Panx1 and Panx2 was mainly found in cell bodies below the sustentacular cell layer in the olfactory epithelium, indicating that Panx1 and Panx2 are expressed in mature and immature olfactory neurons, and basal cells. Expression of Panx2 was observed in sustentacular cells in a few locations of the olfactory epithelium. In the olfactory bulb, Panx1 and Panx2 were expressed in spatial patterns. Many mitral cells, tufted cells, periglomerular cells and granule cells were Panx1 and Panx2 positive. Mitral cells located at the dorsal and lateral portions of the olfactory bulb showed weak Panx1 expression compared with those in the medial side. However, the opposite was true for the distribution of Panx2 positive mitral cells. There were more Panx2 mRNA positive mitral cells and granule cells compared to those expressing Panx1. Our findings on pannexin expression in the olfactory system of adult mice raise the novel possibility that pannexins play a role in information processing in the olfactory system. Demonstration of expression patterns of pannexins in the olfactory system provides an anatomical basis for future functional studies.     

Olfactory epithelia differentially express neuronal markers

All three olfactory epithelia, the olfactory epithelium proper (OE), the septal organ of Masera (SO), and the vomeronasal organ of Jacobson (VNO) originate from the olfactory placode. Here, their diverse neurochemical phenotypes were analyzed using the immunohistochemical expression pattern of different neuronal markers. The olfactory bulb (OB) served as neuronal control. Neuronal Nuclei Marker (NeuN) is neither expressed in sensory neurons in any of the three olfactory epithelia, nor in relay neurons (mitral/tufted cells) of the OB. However, OB interneurons (periglomerular/granule cells) labeled, as did supranuclear structures of VNO supporting cells and VNO glands. Protein Gene Product 9.5 (PGP9.5 = C-terminal ubiquitin hydrolase L1 = UCHL1) expression is exactly the opposite: all olfactory sensory neurons express PGP9.5 as do OB mitral/tufted cells but not interneurons. Neuron Specific Enolase (NSE) expression is highest in the most apically located OE and SO sensory neurons and patchy in VNO. In contrast, the cytoplasm of the most basally located neurons of OE and SO immunoreacted for Growth Associated Protein 43 (GAP-43/B50). In VNO neurons GAP-43 labeling is also nuclear. In the cytoplasm, Olfactory Marker Protein (OMP) is most intensely expressed in SO, followed by OE and least in VNO neurons; further, OMP is also expressed in the nucleus of basally located VNO neurons. OB mitral/tufted cells express OMP at low levels. Neurons closer to respiratory epithelium often expressed a higher level of neuronal markers, suggesting a role of those markers for neuronal protection against take-over. Within the VNO the neurons show clear apical–basal expression diversity, as they do for factors of the signal transduction cascade. Overall, expression patterns of the investigated neuronal markers suggest that OE and SO are more similar to each other than to VNO.     

Tissue- and species-specific expression patterns of class I,III, and IV Adh and Aldh1 mRNAs in rodent embryos

Alcohol and aldehyde dehydrogenases (ADHs and ALDHs) may be of interest in the pathology of Parkinson's disease (PD) because of their role in protection against toxins and in retinoid metabolism, which is required for growth and development of the mesencephalic dopamine system. In the present study, the spatial and temporal expression patterns of Adh1, Adh3, Adh4, and Aldh1 mRNAs in embryonic C57BL/6 mice (E9.5-E19.5) and Sprague-Dawley rats (E12.5-P0) have been investigated by using radioactive oligonucleotide in situ hybridization. High expression of Aldh1 mRNA was found in the developing mesencephalic dopamine neurons of both mice and rats. Expression of Adh1 and Adh4 mRNAs was observed in adrenal cortex and olfactory epithelium in mice. Additionally, Adh1 was expressed in epidermis, liver, conjunctival, and intestinal epithelium. In rat embryos, expression was less extensive, with Adh1 mRNA being found in liver and intestines. Adh3 expression was ubiquitous in both mouse and rat embryos, suggesting a housekeeping function of the gene. Consistent with previous studies in adult rats and mice, our data suggest that Adh3 is the only ADH class present in rodent brain. Adh and Aldh gene activity in mouse and rat embryos indicate the possible involvement of the respective enzymes in retinoid metabolism and participation in defense against toxic insults, including those that may be involved in the pathogenesis of PD. This work was supported by grants from the Swedish Research Council, the Swedish Parkinson Foundation, the Swedish Brain Foundation, Karolinska Institutet funds, AstraZeneca, and the US Public Health Service.     

The expression of proprotein convertase PACE4 is highly regulated by Hash-2 in placenta: possible role of placenta-specific basic helix-loop-helix transcription factor, human achaete-scute homologue-2

PACE4 is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which contribute to the activation of transforming growth factor (TGF) beta family proteins. We previously reported that PACE4 is highly expressed in syncytiotrophoblasts of human placenta [Tsuji et al. (2003) BIOCHIM: Biophys. Acta 1645, 95-104]. In this study, the regulatory mechanism for PACE4 expression in placenta was analyzed using a human placental choriocarcinoma cell line, BeWo cells. Promoter analysis indicated that an E-box cluster (E4-E9) in the 5'-flanking region of the PACE4 gene acts as a negative regulatory element. The binding of human achaete-scute homologue 2 (Hash-2) to the E-box cluster was shown by gel mobility-shift assay. The overexpression of Hash-2 caused a marked decrease in PACE4 gene expression. When BeWo cells were grown under low oxygen (2%) conditions, the expression of Hash-2 decreased, while that of PACE4 increased. In both cases, other SPCs, such as furin, PC5/6, and PC7/8, were not affected. Further, PACE4 expression was found to be developmentally regulated in rat placenta. By in situ hybridization, Mash-2 (mammalian achaete-scute homologue 2) mRNA was found to be expressed in the spongiotrophoblast layer where PACE4 was not expressed. In contrast, the PACE4 mRNA was expressed mainly in the labyrinthine layer where Mash-2 was not detected. These results suggest that PACE4 expression is down-regulated by Hash-2/Mash-2 in both human and rat placenta and that many bioactive proteins might be regulated by PACE4 activity.     

Localization of type I insulin-like growth factor receptor messenger RNA in the adult rat brain by in situ hybridization.

Using multiple 35S-labeled oligonucleotide probes concurrently, the type I insulin-like growth factor receptor (IGF-I-R) mRNA was demonstrated by Northern blot hybridization in newborn and adult rat brain as a single species of approximately 11 kilobases. The probes were used to localize IGF-I-R mRNA by in situ hybridization in slices of adult rat brain. The highest levels of IGF-I-R mRNA expression were found in the glomerular and mitral cell body layers of the olfactory bulb, the granule cell body layers of the dentate gyrus and cerebellum, the pyramidal cell body layers of the piriform cortex and Ammon's horn, and the choroid plexus. The lowest levels of IGF-I-R mRNA expression were found in white matter. At the cellular level, IGF-I-R mRNA was expressed by a variety of neurons, by epithelial cells of the choroid plexus, and by ependymal cells of the third ventricle. Of the neuron types studied, the highest levels of IGF-I-R mRNA were consistently found in perikarya of mitral and tufted cells in the olfactory bulb, in pyramidal cells of the piriform cortex and Ammon's horn, and in granule cells of the dentate gyrus. There was a close congruency between the distribution of IGF-I binding and IGF-I-R mRNA at the regional level. Neuropil layers in the cerebral cortex, olfactory bulb, hippocampus, and cerebellum contained a high level of IGF-I binding, whereas the adjacent cell body layers contained a high level of the IGF-I-R mRNA. We conclude that in these regions, IGF-I-R mRNA is synthesized in neuronal cell bodies, and the receptors are transported to axons and dendrites in adjacent synapse-rich layers, where appropriate IGF effects are achieved.     

Targeted Deletion of the ERK5 MAP Kinase Impairs Neuronal Differentiation,Migration, and Survival during Adult Neurogenesis in the Olfactory Bulb

Recent studies have led to the exciting idea that adult-born neurons in the olfactory bulb (OB) may be critical for complex forms of olfactory behavior in mice. However, signaling mechanisms regulating adult OB neurogenesis are not well defined. We recently reported that extracellular signal-regulated kinase (ERK) 5, a MAP kinase, is specifically expressed in neurogenic regions within the adult brain. This pattern of expression suggests a role for ERK5 in the regulation of adult OB neurogenesis. Indeed, we previously reported that conditional deletion of erk5 in adult neurogenic regions impairs several forms of olfactory behavior in mice. Thus, it is important to understand how ERK5 regulates adult neurogenesis in the OB. Here we present evidence that shRNA suppression of ERK5 in adult neural stem/progenitor cells isolated from the subventricular zone (SVZ) reduces neurogenesis in culture. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, stimulates neurogenesis. Furthermore, inducible and conditional deletion of erk5 specifically in the neurogenic regions of the adult mouse brain interferes with cell cycle exit of neuroblasts, impairs chain migration along the rostral migratory stream and radial migration into the OB. It also inhibits neuronal differentiation and survival. These data suggest that ERK5 regulates multiple aspects of adult OB neurogenesis and provide new insights concerning signaling mechanisms governing adult neurogenesis in the SVZ-OB axis.     

cDNA cloning of putative rat acetyl-CoA transporter and its expression pattern in brain

Rat acetyl-CoA transporter gene (Acatn) encodes a hydrophobic multi-transmembrane protein involved in the O-acetylation of gangliosides. O-acetylated gangliosides have been found to play important roles in the embryonic development of the nervous system. We have isolated rat Acatn cDNA by PCR cloning. The amino acid sequence of rat Acatn exhibited 92% and 96% homology with human and mouse sequences, respectively. The mRNA was expressed in brain at all developmental stages. Acatn expression was higher in embryonic and postnatal rats than in adult rats. Cellular localization of Acatn mRNA in adult rat brain was also analyzed by in situ hybridization. Acatn mRNA expression was detected in the neuronal cells of cerebellum, hippocampus, hypothalamus, cortex, olfactory bulb, and dorsal and ventral anterior olfactory nucleus in adult rat brain.     

Light responsiveness of clock genes, Per1 and Per2, in the olfactory bulb of mice

The olfactory bulb (OB) of rodents has been suggested to possess a self-sustaining circadian oscillator which functions independent from the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, neither histology nor physiology of this extra-SCN clock is studied yet. In the present study, we examined circadian variation of major clock gene expressions in the OB and responsiveness to single photic stimuli. Here we show significant circadian variation in the expression of clock genes, Per1, Per2 and Bmal1 in the OB. Per1 and PER2 were mainly expressed in the mitral cell and granular cell layers of the OB. Light responsiveness of Per1 and Per2 expression was different in the OB from that in the parietal cortex. Both Per1 and Per2 are expressed in the OB only by l000 lux light pulse, whereas 100 lux light was enough to induce Per1 mRNA in the parietal cortex. Interestingly, even 1000 lux light failed to induce Per2 mRNA in the parietal cortex. These clock gene-specific and brain region-dependent responses to lights in the OB and parietal cortex suggest that single light stimulus induces various physiological functions in different brain areas via specific clock gene.     

Cellular expression of MAP 2 kinase in rat brain

The cellular localization of microtubule-associated protein (MAP) 2 kinase mRNA in rat brain was examined by in situ hybridization histochemistry using a synthetic oligonucleotide probe. MAP 2 kinase was expressed in both neuronal and non-neuronal cells. ‘Areas of high density of mRNA label by the MAP 2 kinase probe appeared to be associated with high cellular packing density. Thus, MAP 2 kinase expression was particularly high in regions such as the locus coeruleus, the piriform cortex, the dentate gyrus granule cell layer, pyramidal cells of the hippocampus, the mitral cells of the olfactory bulb, and the large motor neurons of the V and VII nerves. This apparent ubiquitous distribution suggests an important role of MAP 2 kinase in the cellular functions in most cells of the adult brain.     

Expression of Coxsackie-Adenovirus receptor (CAR) in the developing mouse olfactory system

Interest in manipulating gene expression in olfactory sensory neurons (OSNs) has led to the use of adenoviruses (AdV) as gene delivery vectors. OSNs are the first order neurons in the olfactory system and the initial site of odor detection. They are highly susceptible to adenovirus infection although the mechanism is poorly understood. The Coxsackie-Adenovirus receptor (CAR) and members of the integrin family have been implicated in the process of AdV infection in various systems. Multiple serotypes of AdV efficiently bind to the CAR, leading to entry and infection of the host cell by a mechanism that can also involve integrins. Cell lines that do not express CAR are relatively resistant, but not completely immune to AdV infection, suggesting that other mechanisms participate in mediating AdV attachment and entry. Using in situ hybridization and western blot analyses, we show that OSNs and olfactory bulbs (OB) of mice express abundant CAR mRNA at embryonic and neonatal stages, with progressive diminution during postnatal development. By contrast to the olfactory epithelium (OE), CAR mRNA is still present in the adult mouse OB. Furthermore, despite a similar postnatal decline, CAR protein expression in the OE and OB of mice continues into adulthood. Our results suggest that the robust AdV infection observed in the postnatal olfactory system is mediated by CAR and that expression of even small amounts of CAR protein as seen in the adult rodent, permits efficient AdV infection and entry. CAR is an immunoglobulin domain-containing protein that bears homology to cell-adhesion molecules suggesting the possibility that it may participate in organization of the developing olfactory system.