Olfactory epithelium destruction by ZnSO4 modified sulfhydryl oxidase expression in mice

Experimental destruction of olfactory neurons stimulates proliferation and differentiation of local neural precursors and is used as a model to study in vivo mechanisms for degeneration and regeneration of the nervous system. Quiescin-sulfhydryl oxidases (QSOX) have a potential role in the control of the cell cycle or growth regulation and have recently been described in the central nervous system. In mice, we show an expression of QSOX in olfactory mucosa. Northern- and western-blot analysis show that the destruction of olfactory epithelium is associated with a reversible reduction in QSOX expression. Interestingly, QSOX is not localized in olfactory neurons (ON) but in cells of the lamina propria, suggesting that olfactory epithelium destruction may act as a signal of down-regulation of QSOX expression.     

Expression of stathmin and SCG10 proteins in the olfactory neurogenesis during development and after lesion in the adulthood

Stathmin and SCG10 belong to a family of phosphoproteins associated to cell proliferation and differentiation. In the present study, we have analyzed immunocytochemically the distribution of these proteins during neurogenesis in the mouse olfactory system, from midgestation to adulthood. Data show that already at embryonic day 12, stathmin and SCG10 immunoreactivities were present in the olfactory and vomeronasal neurons, and their number increased greatly, colocalizing with neuronal specific tubulin, a marker of immature neurons. Later on up to adulthood, the distribution of stathmin and SCG10 became progressively restricted to a few immature receptor and chemosensory neurons. Significantly, in the olfactory epithelium, stathmin was seen in immature neurons and also in basal cells representing precursors of neuronal elements. Interestingly, before birth stathmin and SCG10 immunopositive cells were seen outside the olfactory epithelium, seemingly migrating toward the olfactory bulb. After regeneration in the adult following peripheral lesion of the olfactory epithelium, stathmin and SCG10 were again strongly expressed and generally colocalized with neuronal specific tubulin immunoreactivity. Overall these results indicate that stathmin and SCG10 are expressed in immature olfactory neurons as well as in the migrating cells generated from the olfactory epithelium, supporting the role of these proteins in neurogenesis and cell migration.     

Regulation of olfactory neurogenesis by amidated neuropeptides

The existence of stem cells in the CNS raises issues concerning the ability of nervous tissues to regenerate in the adult mammal and provides new perspectives on the treatment of degenerative disease and traumatic injury of the nervous system. These cells have a relatively limited range of locations within the nervous system and include cells of the rostral migratory stream, hippocampus, retina, and olfactory epithelium. The olfactory epithelium has been studied as a model of adult neuronal regeneration, with neuronal precursor/basal cells serving as the olfactory "stem cells." The identification of factors that promote neuronal proliferation or regeneration within the olfactory epithelium can provide clues to the process of adult mammalian nervous system repair and treatment. Multiple factors have been examined that appear to influence the proliferation and subsequent maturation of basal cells. These factors include nerve growth factor, fibroblast growth factor-2, epidermal growth factor, and insulin/insulin-like growth factor-1. Recently, two amidated neuropeptides, neuropeptide Y (NPY) and pituitary adenylate cyclase-activating polypeptide (PACAP38), identified in the olfactory epithelium have been shown to promote dramatically neuronal proliferation. The effects of NPY and PACAP suggest that amidated neuropeptides may serve a broad developmental and regenerative role in the mammalian olfactory epithelium.     

Matrix metalloproteinase expression in the olfactory epithelium

The olfactory epithelium contains neuronal progenitor cells capable of continuous neurogenesis and is a unique model for studying neural degeneration, regeneration, axon outgrowth and recovery from injury. Matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs), have been implicated in cell turnover, development, migration, and metastatic processes. We used Western blot and immunohistochemistry to determine whether MMP-2 and associated proteins TIMP-2 and membrane type 1 matrix metalloproteinase (MT1-MMP) are present in the olfactory epithelium of mice. We found MMP-2 expression localized to the olfactory basal cells and immature neurons. After injury-induced neural degeneration, MMP-2 and MT1-MMP levels decreased while TIMP-2 levels increased. However, following 35 days of neurogenesis and cell replacement TIMP-2 and MT1-MMP returned to control levels. The results show a correlation between MMP and TIMP levels and the stages of neural degeneration, regeneration and recovery of the olfactory epithelium following injury.     

Rat olfactory cells and a central nervous system neuronal subpopulation share a cell surface antigen

Using the monoclonal antibody (Mab) 6B7, a cell surface component found in adult rat central nervous system membrane preparations and on the surfaces of a subpopulation of neurons in cultures of embryonic rat forebrain has been identified. This Mab was derived from mice immunized with a rat forebrain synaptic plasma membrane preparation. High levels of Mab 6B7 binding are observed with membrane preparations from rat forebrain and olfactory bulb but no detectable binding is observed with membranes from the non-neural adult rat tissues heart, kidney, liver, lung and testes. Binding to dorsal root ganglia preparations was 5-fold lower than to forebrain. In immunofluorescence analyses, Mab 6B7 binds to the surface of a significant proportion of neurons in cultures of embryonic day 14 rat forebrain. However, it is absent from GFAP-positive astrocytes, oligodendrocytes, Schwann cells and fibroblastic cells in rat neural cultures. Due to the high levels of binding in olfactory tissue, the distribution of the 6B7 antigen in the olfactory epithelium was characterized in greater detail. In cryostat sections, 6B7 appears to react with a cell population of the basal layer of the adult rat epithelium, but is absent from the cell bodies of the more mature neuronal population which lies higher in the epithelium. This result suggests that within the olfactory epithelium Mab 6B7 may be useful as a marker for the proliferative basal cells which are the neuronal precursors in the epithelium. In summary, the 6B7 antigen may be useful in identifying and analyzing cell subpopulations in both the central nervous system and olfactory epithelium.     

A new model for olfactory placode development

The peripheral nervous system of vertebrate animals arises primarily from the interaction of cranial neural crest and sensory placodes. Placodes are described as thickenings of ectoderm that arise through cell division during neural tube formation. The olfactory sensory system is one component of the peripheral nervous system that arises from paired sensory placodes during development. The olfactory placodes give rise to the primary sensory neurons, support cells and basal cells of the olfactory epithelium. Recent evidence from work in zebrafish and chick suggests that the olfactory and auditory placodes arise from large fields of cells that converge to form the sensory placode. The olfactory placodes arise from within the neural plate, and cell division is apparent only after the sensory placodes are morphologically distinct. As the olfactory placode is forming, its precursors must segregate from their neighboring fields which will give rise to the adenohypophyseal placode, cranial neural crest, and telencephalon. Analysis has shown that the endocrine cells thought to arise from the olfactory placode originate in the neighboring adenohypophyseal and cranial neural crest domains. The borders separating the domains are plastic, where cells sort as they move, and cell fate is dependent on the identity of neighbors once the cells have converged to form the sensory placode. Thus there is degeneracy built into the system such that cells accommodate changes in the environment until cell migrations controlling the formation of the sensory placodes are complete.     

Association of ecto-5′-nucleotidase with specific cell types in the adult and developing rat olfactory organ

A unique feature of the olfactory epithelium is its ability to give rise to new sensory neurons throughout life and also following injury. Cells at the basal side of the epithelium serve as neurogenic progenitor cells. The enzyme ecto-5′-nucleotidase is expressed at the surface of developing nerve cells and is regarded as a marker of neural development. To study the expression pattern of the enzyme, we analyzed its distribution in the adult and developing rat olfactory organ. Labeling is restricted to specific cell types and varies between the epithelia investigated. At the basal side of the olfactory epithelium, activity of 5′-nucleotidase is associated specifically with the dark/horizontal basal cells. Neither the light/globose basal cells, which are the immediate precursors of the sensory receptor cells, nor subsets of potentially immature olfactory receptor cells are labeled. On the other hand, microvillar cells dispersed at the lumenal side of the epithelium contain 5′-nucleotidase activity. The enzyme is also present at the inner lining of the ducts of Bowman's glands as they traverse the epithelium. Within the respiratory epithelium, activity of 5′-nucleotidase is associated with basal cells as well as with the epithelial surface. During development, 5′-nucleotidase is initially limited to the respiratory epithelium, including its basal cells. Dark/horizontal basal cells of the olfactory epithelium, which are positive for 5′-nucleotidase, first appear at the border of the respiratory epithelium, suggesting that they might originate from immigrating basal cells of the respiratory epithelium. Within the vomeronasal organ, labeling is largely restricted to the receptor-free epithelium. Although the functional role of 5′-nucleotidase in the olfactory system needs to be further defined, the distribution of the enzyme can be used successfully as a marker for defined cell types. J. Comp. Neurol. 393:528–537, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Characterization of potential precursor populations in the mouse olfactory epithelium using immunocytochemistry and autoradiography.

There are at least two basal cell populations in the olfactory epithelium that could give rise to olfactory neurons during development, in the normal adult, and after experimentally induced receptor cell death. These populations have been subdivided as horizontal (HBC) and globose (GBC) basal cells on the basis of morphological criteria and by staining with antibodies against cytokeratin. HBCs are positive for cytokeratin while GBCs are negative. We have studied which cell type is induced to divide during receptor cell regeneration stimulated by olfactory bulbectomy using a combination of immunocytochemistry and autoradiography. By examining which population increases its labeling index with 3H-thymidine (3H-TdR) at various times after bulbectomy, it is shown that there is an increase in 3H-TdR uptake in the cytokeratin-negative GBCs with no change in the cytokeratin-positive HBCs. This suggests that the GBCs are specifically induced to divide in response to cues that accompany receptor cell death, and it is thus concluded that these cells are among the precursors of new olfactory receptor neurons.     

Immunochemical markers for the frog olfactory neuroepithelium

Monoclonal antibodies (mAbs) were generated that react with the major cell types in the olfactory neuroepithelium of the frog, Rana catesbeiana. This pseudostratified epithelium consists of apical supporting cells, a middle layer of olfactory receptor neurons and a heterogeneous population of basal cells consisting of basal cells proper and globose basal cells. Both olfactory receptor neurons and globose basal cells were labelled by mAb 13-OE, which recognized the neural cell adhesion molecule NCAM. The identity of these NCAM positive cells was established by analysing regenerating olfactory epithelium and by a double-antibody labelling immunofluorescence technique. The olfactory nerve was lesioned, which induced the death of olfactory receptor neurons and the subsequent proliferation of basal cells. When the regenerating olfactory epithelium was analysed prior to the reconstitution of mature olfactory neurons, mAb 13-OE reacted specifically with globose basal cells and not the basal cells proper. Simultaneous labelling of normal olfactory epithelium with mAb 13-OE and polyclonal anti-keratin antibodies, the latter of which labels supporting cells and basal cells proper, revealed no double-labelled cells. These results further confirmed that NCAM was expressed by both globose basal cells and receptor neurons but not by other cell types within the epithelium. Additional cell types in the olfactory epithelium reacted with other new mAbs: 4-OE, 5-OE, 7-OE and 9-OE. Supporting cells were stained by mAb 4-OE. Olfactory receptor neurons and the entire population of basal cells were immunoreactive with mAb 7-OE. The cilia and knobs of receptor neurons were strongly immunoreactive with mAb 5-OE whereas mAb 9-OE selectively stained olfactory knobs and not the cilia on these chemosensory cells. These studies are a first step towards experimental approaches designed to elucidate the mechanisms underlying the unique proliferative properties of the olfactory neuroepithelium in frog.     

Hypothyroidism disrupts neural development in the olfactory epithelium of adult mice

Adult mice made hypothyroid with propylthiouracil (PTU) lose their sense of smell. This is prevented by daily administration of thyroxine. As thyroxine is necessary for the correct development of the nervous system it may also be necessary for the genesis of new olfactory receptor neurones, a process that continues into adulthood. Adult mice were treated with PTU, injected with [3H]thymidine after 54 days and killed 5 or 15 days later. Microscopic analysis of the olfactory epithelium after autoradiography revealed similar numbers of labelled nuclei in the basal cell layer of the olfactory epithelia of Control and Hypothyroid mice 5 days after injection with [3H]thymidine. This indicated similar rates of basal cell division in the two groups. Fifteen days after [3H]thymidine injection, however, there were fewer labelled nuclei in the receptor cell layer of Hypothyroid mice and the olfactory epithelium was thinner than in Controls. Thyroxine therapy which reversed PTU-induced anosmia also reversed the epithelial effects of PTU treatment. Somewhat unexpectedly, there were no differences between the treatment groups in the average diameter of glomeruli in the olfactory bulb, and no differences in the expression of olfactory marker protein. The results indicate that although hypothyroidism disrupts neural development in the olfactory epithelium, it does not lead to a complete loss of mature receptor neurones.     

Age-dependent effect of nitric oxide on subventricular zone and olfactory bulb neural precursor proliferation

Nitric oxide (NO) synthase (NOS) is developmentally regulated in the embryonic brain, where NO participates in cell proliferation, survival, and differentiation. In adults, NO inhibits neurogenesis under physiological conditions. This work investigates whether the NO action is preserved all along development up to adulthood or whether its effects in adults are a new feature acquired during brain maturation. The relationship between nitrergic neurons and precursors, as well as the functional consequences of pharmacological NOS inhibition, were comparatively analyzed in the subventricular zone (SVZ) and olfactory bulb (OB) of postnatal (P7) and adult (>P60) mouse brains. The SVZ was markedly reduced between P7 and adults, and, at both ages, neurons expressing neuronal NOS (nNOS) were found in its striatal limits. In postnatal mice, these nitrergic neurons contained PSA-NCAM, and their projections were scarce, whereas, in adults, mature nitrergic neurons, devoid of PSA-NCAM, presented abundant neuropil. In the OB, local proliferation almost disappeared in the transition to adulthood, and periglomerular nitrergic neurons, some of which were PSA-NCAM positive, were found in postnatal and adult mice. Administration of the NOS inhibitor L-NAME did not affect cell proliferation in the SVZ or in the OB of postnatal mice, whereas it significantly enhanced the number of mitotic cells in both regions in adults. Thus, the NO action on SVZ neurogenesis is a phenomenon that appears after the postnatal age, which is probably due to the germinal layer size reduction, allowing exposure of the NO-sensitive neural precursors to the NO produced in the SVZ-striatum limits.     

5HTR3A‐driven GFP labels immature olfactory sensory neurons

The ionotropic serotonin receptor, 5‐HT₃, is expressed by many developing neurons within the central nervous system. Since the olfactory epithelium continues to generate new olfactory sensory neurons (OSNs) throughout life, we investigated the possibility that 5‐HT₃ is expressed in the adult epithelium. Using a transgenic mouse in which the promoter for the 5‐HT_3a subunit drives expression of green fluorescent protein (GFP), we assessed the expression of this marker in the olfactory epithelium of adult mice. Both the native 5‐HT_3a mRNA and GFP are expressed within globose basal cells of the olfactory and vomeronasal epithelium in adult mice. Whereas the 5‐HT_3a mRNA disappears relatively quickly after final cell division, the GFP label persists for about 5 days, thereby labeling immature OSNs in both the main olfactory system and vomeronasal organ. The GFP‐labeled cells include both proliferative globose basal cells as well as immature OSNs exhibiting the hallmarks of ongoing differentiation including GAP43, PGP9.5, but the absence of olfactory marker protein. Some of the GFP‐labeled OSNs show characteristics of more mature yet still developing OSNs including the presence of cilia extending from the apical knob and expression of NaV1.5, a component of the transduction cascade. These findings suggest that 5‐HT_3a is indicative of a proliferative or developmental state, regardless of age, and that the 5‐HT_3AGFP mice may prove useful for future studies of neurogenesis in the olfactory epithelium. J. Comp. Neurol. 525:1743–1755, 2017. © 2016 Wiley Periodicals, Inc.     

Neuropeptide Y stimulates proliferation, migration and differentiation of neural precursors from the subventricular zone in adult mice

The neuropeptide Y (NPY) is widely expressed in the central nervous system and has been shown to stimulate neurogenesis in the hippocampus and the olfactory epithelium. Here, we demonstrate that intracerebroventricular injection of NPY stimulates proliferation of neural precursors in the mice subventricular zone (SVZ), one the most neurogenic areas of the brain. Newly generated neuroblasts migrate through the rostral migratory stream to the olfactory bulb and also directly to the striatum, as evidenced by BrdU labelling and cell phenotyping. Using knock-out mice, specific NPY receptor agonists and antagonists, we report that this neuroproliferative effect is mediated by the Y1 receptor subtype that we found to be highly expressed in the SVZ both at the mRNA and protein levels. Our data suggest that stimulating endogenous SVZ neural stem cells by NPY may be of a potential interest in cell replacement based therapies of neurodegenerative diseases affecting the striatum such as Huntington's disease.     

Smad3 Deficiency Reduces Neurogenesis in Adult Mice

Transforming growth factor-β signaling through Smad3 inhibits cell proliferation in many cell types. As cell proliferation in the brain is an integral part of neurogenesis, we sought to determine the role of Smad3 in adult neurogenesis through examining processes and structures important to neurogenesis in adult Smad3 null mice. We find that there are fewer proliferating cells in neurogenic regions of adult Smad3 null mouse brains and reduced migration of neuronal precursor cells from the subventricular zone to the olfactory bulb. Alterations in astrocyte number and distribution within the rostral migratory stream of Smad3 null mice give rise to a smaller and more disorganized structure that may impact on neuronal precursor cell migration. However, the proportion of proliferating cells that become neurons is similar in wild type and Smad3 null mice. Our results suggest that signaling through Smad3 is needed to maintain the rate of cell division of neuronal precursors in the adult brain and hence the amount of neurogenesis, without altering neuronal cell fate.     

CX₃CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX?CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX?CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX?CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX?CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX?CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX?CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX?CL1. Collectively, our data indicate that signaling through the CX?CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.     

Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells

We have infused replication-incompetent retroviral vectors into the nasal cavity of adult rats 1 day after exposure to the olfactotoxic gas methyl bromide (MeBr) to assess the lineage relationships of cells in the regenerating olfactory epithelium. The vast majority of the retrovirus-labeled clones fall into three broad categories: clones that invariably contain globose basal cells (GBCs) and/or neurons, clones that always include cells in the ducts of Bowman's glands, and clones that are composed of sustentacular cells only. Many of the GBC-related clones contain sustentacular cells and horizontal basal cells as well. Most of the duct-related clones contain gland cells, and some also include sustentacular cells. Thus, the destruction of both neurons and non-neuronal cells that is caused by MeBr activates two distinct types of multipotent cells. The multipotent progenitor that gives rise to neurons and non-neuronal cells is a basal cell, whereas the progenitor that gives rise to duct, gland, and sustentacular cells resides within the ducts, based on the pattern of sparing after lesion and the analysis of early regeneration by using cell type-specific markers. We conclude that the balance between multipotency and selective neuropotency, which is characteristic of globose basal cells in the normal olfactory epithelium, is determined by which cell types have been depleted and need to be replenished rapidly. J. Comp. Neurol. 400:469–486, 1998. © 1998 Wiley-Liss, Inc.