Neuronal degeneration and regeneration in the olfactory epithelium of pigeon following transection of the first cranial nerve.

The pigeon olfactory nerve has been sectioned to explore the course of retrograde degeneration of the sensory neurons' perikarya, which are located in the olfactory neuroepithelium. Both light- and electron-microscopic observations have shown that from 3 to 8 days after axotomy the sensory neurons undergo retrograde, irreversible degeneration. Following disappearance of the mature neurons, the basal cells of the neuroepithelium actively divide and differentiate into mature olfactory sensory neurons. Consequently, the basal cells represent true stem cells of the olfactory sensory neurons. The olfactory mucosa regains a structural organization close to normal in a period of 30-50 days after axotomy. These observations indicate that, when the primary olfactory neurons degenerate as a consequence of the experimental section of their axons, restitutio ad integrum of the sensory olfactory connections can be reestablished by new elements which differentiate from basal cells of the olfactory neuroepithelium.20     

Expression of mouse beta 2-microglobulin in frozen and formaldehyde-fixed central nervous tissues: comparison of tissue behind the blood-brain barrier and tissue in a barrier-free region

Previous work indicates that the weak expression in neural tissues of beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I gene products can be increased experimentally. Physiologic conditions in which greater neural MHC expression occurs are not well defined. Here we have asked whether protection from blood-borne antigens afforded by the blood-brain barrier is related to the lack of MHC expression. A rabbit antiserum raised against purified mouse beta 2-m was used in an immunocytochemical assay. The serum reacted strongly with lymphoid tissues and was inhibited by purified beta 2-m. No beta 2-m was detected in neurons or glia in any brain area examined. A barrier-free region, the area postrema, showed the same lack of neural cell staining. Blood vessel walls in the same sections were beta 2-m+. It is unlikely that these staining patterns are due to cell type-specific beta 2-m degradation, since frozen and formaldehyde-perfused, paraffin-embedded preparations gave similar results. Failure to detect beta 2-m in the area postrema suggests that passive exposure to environmental antigens, immunomodulators, or immunocompetent cells is not sufficient to induce neural class I expression. Rather, if increased expression of beta 2-m and class I occurs in vivo, additional stimulus is required.     

Extraepithelial cells expressing distinct olfactory receptors are associated with axons of sensory cells with the same receptor type

During critical phases of mouse development, axons from olfactory sensory neurons grow out of the nasal neuroepithelium and navigate through the connective mesenchyme tissue towards their targets in the developing telencephalic vesicle. Between embryonic days E11 and E16, populations of cells are located in the mesenchyme which express distinct olfactory receptor genes along with the olfactory marker protein (OMP); thus they express markers characteristic for mature olfactory sensory neurons. These extraepithelial cells are positioned along the axon tracts, and each population expressing a given receptor gene is specifically associated with the axons of those olfactory sensory neurons with the same receptor type. The data suggest that they either might be guide posts for the outgrowing axons or migrate along the axons into the brain.     

Specific molecular interaction between the insulin receptor and a D product of MHC class I

The density of MHC class I was determined on a murine thymoma cell line (R1), an H-2 negative variant (R1E), and R1E-derived cell lines in which H-2 expression was restored by transfection of various MHC class I genes (Db, Kb, and truncated Db) and/or a beta-2-microglobulin gene (beta 2-m; B2). Appreciable MHC class I expression was found on R1 cells and on the variants in which MHC class I expression was restored by transfection of Db/beta 2-m or Kb/beta 2-m genes. Only approximately 20% difference was observed between the number of Db molecules and Kb molecules on the R1E/B2/Db and on R1E/B2/Kb, respectively. However, specific insulin binding was significantly different between these lines. By using a computer assisted curve fitting program, the insulin binding data for R1 and R1E/B2/Db cell lines best fitted a two-site model (K approximately 6 x 10(-9) M for high-affinity sites and a 2 to 3 x 10(-7) M for low-affinity sites), whereas all other lines only expressed one type of insulin binding site. These sites were unrelated to IGF-I and IGF-II receptors. Cross-linking of 125I-labeled insulin demonstrated specific binding of the ligand to a Mr approximately 130,000 dalton band in all lines. In the R1E/B2/Db cells, insulin also cross-linked to cell membrane molecules with Mr approximately 48,000 and approximately 60,000 Da, which were identified by immunoprecipitation to be the H chain of MHC class I and the heavy chain of MHC class I plus beta 2-m, respectively. It is concluded that the insulin receptors in the cell membrane interact specifically with D-products of MHC class I and that class I molecules of MHC may have a crucial role in insulin receptor expression. This may reflect a more general nonimmunologic role of MHC class I.     

Differential synthesis of β-tubulin isotypes in gerbil nasal epithelia

Compartmentalization of beta-tubulin isotypes within cells according to function was examined in gerbil olfactory and respiratory epithelia by using specific antibodies to four beta-tubulin isotypes (beta(I), beta(II), beta(III), and beta(IV)). Isotype synthesis was cell-type-specific, but the localization of the isotypes was not compartmentalized. All four isotypes were found in the cilia, dendrites, somata, and axons of olfactory neurons. Only two isotypes (beta(I) and beta(IV)) were present in the cilia of nasal respiratory epithelial cells. The beta(IV) isotype, thought to be an essential component of cilia, was present in olfactory neurons and respiratory epithelial cells, which are ciliated, but was not found in basal cells (the stem cells of olfactory sensory neurons, which have no cilia). Olfactory neurons therefore do not synthesize beta(IV)-tubulin until they mature, when functioning cilia are also elaborated. The failure to observe compartmentalization of beta-tubulin isotypes in olfactory neurons sheds new light on potential functions of the beta-tubulin isotypes.     

Olfactory receptor gene expression

Recognition and discrimination of odorous molecules are determined by heptahelical G-protein-coupled receptor proteins localized primarily in the ciliary membrane of olfactory sensory neurons. The discovery of a large multigene family encoding odorant receptors allows us to approach various facets concerning the molecular basis of olfactory chemospecificity, ranging from chromosomal localization and control of expression of olfactory receptor genes to temporal and spatial expression patterns of various receptor types in the nasal neuroepithelium. The target-independent onset of receptor expression and its topographical organization suggest a precommited functional identity of olfactory neurons.     

Axon mis-targeting in the olfactory bulb during regeneration of olfactory neuroepithelium

During development, primary olfactory axons typically grow to their topographically correct target zone without extensive remodelling. Similarly, in adults, new axons arising from the normal turnover of sensory neurons essentially project to their target without error. In the present study we have examined axon targeting in the olfactory pathway following extensive chemical ablation of the olfactory neuroepithelium in the P2-tau:LacZ line of mice. These mice express LacZ in the P2 subpopulation of primary olfactory neurons whose axons target topographically fixed glomeruli on the medial and lateral surfaces of the olfactory bulb. Intraperitoneal injections of dichlobenil selectively destroyed the sensory neuroepithelium of the nasal cavity without direct physical insult to the olfactory neuron pathway. Primary olfactory neurons regenerated and LacZ staining revealed the trajectory of the P2 axons. Rather than project solely to their topographically appropriate glomeruli, the regenerating P2 axons now terminated in numerous inappropriate glomeruli which were widely dispersed over the olfactory bulb. While these errors in targeting were refined over time, there was still considerable mis-targeting after four months of regeneration.     

An ultrastructural and immunohistological study of the rat olfactory epithelium: Unique properties of olfactory sensory cells

The olfactory epithelium contains three cell types: basal cells, supporting cells and sensory neurons. Electron microscopy as well as immunofluorescence microscopy with intermediate-filament antibodies were used to study the rat olfactory epithelium in order to obtain more information about these different cell types and to try to investigate their histogenetic origins. We found mitoses in the basal-cell layer, as well as multiple centrioles and tonofilaments in some basal cells. As revealed by electron microscopy, the supporting cells contained tonofilaments and reacted strongly with antibodies to keratin, in line with their known epithelial nature. When antibodies to other intermediate-filament types were used, i.e. glial fibrillary acidic protein, vimentin, desmin and neurofilaments, no reaction was seen in the cells of the olfactory epithelium, with the exception of occasional staining of a few axons in the subepithelial layer by neurofilament antibodies. In particular, the cell bodies, dendrites and most axons of the sensory neurons were negative for a variety of antibodies against neurofilaments. Olfactory sensory neurons therefore belong to the very few cells in adult animals which seem to lack intermediate filaments. We discuss whether this finding is related to the fact that these cells are also unique among neurons in that they are not permanent cells but constantly turn over.     

Acquisition of HLA class I W6/32 defined antigenic determinant by heavy chains from different species following association with bovine beta 2-microglobulin

Murine, rat, rabbit and guinea pig class I heavy chains, which do not react with W6/32 monoclonal antibody when they are expressed in association with autologous beta 2-microglobulin (beta 2-m), can acquire such a reactivity once they are expressed at the surface of cells cultured in conditions which allow their association with bovine beta 2-m. Sequence comparison of beta 2-ms suggests that glutamine at position 89 might be critical for the induction of the W6/32 defined antigenic determinant. However, in the murine species, certain class I heavy chains, in spite of their association with bovine beta 2-m, do not express this determinant. Using genetically engineered hybrid class I molecules and selected congenic strains of mice this negative property was shown to be related to the presence of a cysteine residue at position 121 which allows covalent association of beta 2-m to class I heavy chains (Bushkin, Y., J-S. Tung, A. Pinter, J. Michaelson, and E. A. Boyse. 1986. Unusual association of beta 2-microglobulin with certain class I heavy chains of the murine major histocompatibility complex. Proc. Natl. Acad. Sci. USA 83:432). Therefore, expression of the W6/32 defined antigenic determinant implicates both the beta 2-m and the second domain of the heavy chain, but its expression (or exposure) is prevented by the covalent fixation on cysteine 121 of the light chain.     

Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb

In early rat embryos when axons from sensory neurons first contact the olfactory bulb primordium, lactosamine-containing glycans (LCG) are detected on neurons that are broadly distributed within the olfactory epithelium, but that project axons to a very restricted region of the ventromedial olfactory bulb. LCG(+) axons extend through channels defined by the coexpression of galectin-1 and beta2-laminin. These two extracellular matrix molecules are differentially expressed, along with semaphorin 3A, by subsets of ensheathing cells in the ventral nerve layer of the olfactory bulb. The overlapping expression of these molecules creates an axon-sorting domain that is capable of promoting and repelling subsets of olfactory axons. Specifically, LCG(+) axons preferentially grow into the region of the nerve layer that expresses high amounts of galectin-1, beta2-laminin, and semaphorin 3A, whereas neuropilin-1(+) axons grow in a complementary pattern, avoiding the ventral nerve layer and projecting medially and laterally. These studies suggest that initial patterning of olfactory epithelium to olfactory bulb connections is, in part, dependent on extracellular components of the embryonic nerve layer that mediate convergence and divergence of specific axon subsets.     

Immunohistochemical expression of two members of the GDNF family of growth factors and their receptors in the olfactory system

The glial cell line-derived (GDNF) family of trophic factors, GDNF, neurturin, persephin and artemin, are known to support the survival and regulate differentiation of many neuronal populations, including peripheral autonomic, enteric and sensory neurons. Members of this family of related ligands bind to specific GDNF family receptor (GFR) proteins, which complex and signal through the Ret receptor tyrosine kinase. We showed previously that GDNF protein was detectable in olfactory sensory neurons (OSNs) in the olfactory neuroepithelium (ON). In this immunohistochemical study, we localized GDNF, neurturin, GFRα1, GFRα2 and Ret in the adult rat ON and olfactory bulb. We found that GDNF and Ret were widely expressed by immature and mature OSNs, while neurturin was selectively expressed in a subpopulation of OSNs zonally restricted in the ON. The GFRs had differential expression, with mature OSNs and their axons preferentially expressing GFRα1, whereas progenitors and immature neurons more avidly expressed GFRα2. In the bulb, GDNF was highly expressed by the mitral and tufted cells, and by periglomerular cells, and its distribution generally resembled that of Ret, with the exception that Ret was far more predominant on fibers than cell bodies. Neurturin, in contrast, was present at lower levels and was more restricted in its expression to the axonal compartment. GFRα2 appeared to be the dominant accessory protein in the bulb. These data are supportive of two members of this neurotrophic family, GDNF and neurturin, playing different physiological roles in the olfactory neuronal system.     

The low rate of HLA class I molecules on the human embryonic stem cell line HS293 is associated with the APM components' expression level

Human embryonic stem cells (hESCs) represent a promise for future strategies of tissue replacement. However, there are different issues that should be resolved before these cells can be used in cellular therapies; among others, the rejection of transplantable hESCs as a result of HLA incompatibility between donor cells and recipients. The hESCs exhibit a weak HLA class I expression on the cell surface, but today the responsible mechanisms are unknown. We have analyzed the level expression of HLA class I heavy chain, beta2-microglobulin (beta2-m), and antigen-processing machinery (APM) components (TAP1, TAP2, LMP2, LMP7, and Tapasin) using the HS293 hESC line by real-time quantitative RT-PCR. This analysis has revealed a low expression of beta2-m, HLA-B, and Tapasin, and an absence of expression of: TAP1, TAP2, LMP2, and LMP7 genes in the HS293 hESC line respect to the embryoid bodies (EBs) and the induced stem cells with IFNgamma (with significant differences, p<0.05). The lack or loss of HLA class I molecules due to the down-regulation of the APM components has been frequently found in tumors of different histology as specific mechanisms of immune-evasion. We described for the first time in this report that the hESCs shared similar mechanisms with respect to tumor cells responsible for the weak HLA class I expression on the cell surface.     

Defective human leukocyte antigen class I-associated antigen presentation caused by a novel beta2-microglobulin loss-of-function in melanoma cells

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.     

Primary sensory neurons containing command neuron peptide constitute a morphologically distinct class of sensory neurons in the terrestrial snail

In the central nervous system of the terrestrial snail Helix, the gene HCS2, which encodes several neuropeptides of the CNP (command neuron peptide) family, is mostly expressed in cells related to withdrawal behavior. In the present work, we demonstrate that a small percentage (0.1%) of the sensory cells, located in the sensory pad and in the surrounding epithelial region ("collar") of the anterior and posterior tentacles, is immunoreactive to antisera raised against the neuropeptides CNP2 and CNP4, encoded by the HCS2 gene. No CNP-like-immunoreactive neurons have been detected among the tentacular ganglionic interneurons. The CNP-like-immunoreactive fiber bundles enter the cerebral ganglia within the nerves of the tentacles (tentacular nerve and medial lip nerve) and innervate the metacerebral lobe, viz., the integrative brain region well-known as the target area for many cerebral ganglia nerves. The procerebral lobe, which is involved in the processing of olfactory information, is not CNP-immunoreactive. Our data suggest that the sensory cells, which contain the CNP neuropeptides, belong to a class of sensory neurons with a specific function, presumably involved in the withdrawal behavior of the snail.     

Cell surface expression of H2 antigens on primary sensory neurons in response to acute but not latent herpes simplex virus infection in vivo.

CD8(+) T lymphocytes and class I major histocompatibility complex (MHC-I) molecules profoundly influence the severity of neuronal herpes simplex virus (HSV) infection in experimentally infected mice. Paradoxically, neurons are classically regarded as MHC-I deficient. However, it is shown here that H2-encoded heavy chains (alphaCs) and their associated light chain, beta2 microglobulin, are present on the surfaces of primary sensory neurons recovered from sensory ganglia within 1 to 2 weeks of HSV infection. During this time, some neurons are found to be tightly associated with T cells in vivo. Prior data showed that termination of productive HSV infection in the peripheral nervous system is not dependent on cell-mediated lysis of infected neurons. Consistent with these data, immunogold electron microscopy showed that the density of cell surface H2 on neurons is an order of magnitude lower than on satellite glia, which is predicted to favor a noncytolytic CD8 cell response.     

Negatively calcium-modulated membrane guanylate cyclase signaling system in the rat olfactory bulb

The mechanism by which the individual odor signals are translated into the perception of smell in the brain is unknown. The signal processing occurs in the olfactory system which has three major components: olfactory neuroepithelium, olfactory bulb, and olfactory cortex. The neuroepithelial layer is composed of ciliated sensory neurons interspersed among supportive cells. The sensory neurons are the sites of odor transduction, a process that converts the odor signal into an electrical signal. The electrical signal is subsequently received by the neurons of the olfactory bulb, which process the signal and then relay it to the olfactory cortex in the brain. Apart from information about certain biochemical steps of odor transduction, there is almost no knowledge about the means by which the olfactory bulb and cortical neurons process this information. Through biochemical, functional, and immunohistochemical approaches, this study shows the presence of a Ca(2+)-modulated membrane guanylate cyclase (mGC) transduction system in the bulb portion of the olfactory system. The mGC is ROS-GC1. This is coexpressed with its specific modulator, guanylate cyclase activating protein type 1 (GCAP1), in the mitral cells. Thus, a new facet of the Ca(2+)-modulated GCAP1--ROS-GC1 signaling system, which, until now, was believed to be unique to phototransduction, has been revealed. The findings suggest a novel role for this system in the polarization and depolarization phenomena of mitral cells and also contradict the existing belief that no mGC besides GC-D exists in the olfactory neurons.     

Structure and expression of the mouse beta 2-microglobulin gene isolated from somatic and non-expressing teratocarcinoma cells.

Mouse teratocarcinoma cells express neither H-2 heavy chains nor beta 2-microglobulin (beta 2-m). We have constructed two genomic libraries, one from PCC4-aza-RI embryonal carcinoma cells and the other from their adult syngenic counterpart 129/Sv liver cells (H-2bc). The libraries were screened with a full length mouse beta 2-m cDNA probe which we isolated and sequenced. Two cosmid clones carrying the entire beta 2-m gene were isolated, one from each library. There was no detectable difference in structure between the two genes. Furthermore, both were shown to be active and to restore beta 2-m synthesis upon transfer into mutant cells deficient in beta 2-m. Irreversible DNA alterations in or around the beta 2-m gene are thus unlikely to account for the lack of beta 2-m gene expression in embryonal teratocarcinoma cells.     

A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.

The mammalian olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants presumably results from the association of odorous ligands with specific receptors on olfactory sensory neurons. To address the problem of olfactory perception at a molecular level, we have cloned and characterized 18 different members of an extremely large multigene family that encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family are likely to encode a diverse family of odorant receptors.     

The Expression Pattern of Classical MHC Class I Molecules in the Development of Mouse Central Nervous System

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D^b mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D^b in P15 mice. At E10.5, the earliest stage we examined, H2-D^b was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D^b expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D^b mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D^b in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K^b signal paralleled that of H2-D^b and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D^b at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D^b in the developing CNS.