Mary Grillo

The regeneration of the olfactory neuroepithelium following olfactory bulbectomy or peripheral deafferentation was studied with mRNA probes and antibodies for B-50/GAP43 and for olfactory marker protein (OMP). Two stages in the regeneration of the olfactory epithelium could be discerned with these reagents. The first stage occurs following either peripheral deafferentation of the olfactory epithelium with Triton X-100 (TX-100) or after bulbectomy and is characterized by the formation of a large population of immature olfactory receptor neurons. These newly formed neurons express B-50/GAP43, a phosphoprotein related to neuronal growth and plasticity. During the second stage of the regeneration process the newly formed olfactory neurons mature, as evidenced by a decrease in their expression of B-50/GAP43 and an increase in the expression of OMP. This stage is only manifested if the developing neurons have access to the target olfactory bulb. Formation of a full complement of OMP-expressing neurons occurs only after peripheral lesion with TX-100. In contrast, following bulbectomy the reconstituted olfactory epithelium lacks its normal target and is compromised in its ability to recover from nerve damage, as evidenced by the presence of a large number of B-50/GAP43-expressing neurons up to 3 months after the lesion and its failure to establish a full complement of OMP-expressing neurons. These results demonstrate that the olfactory epithelium is capable of replacing its sensory neurons independently of the presence of its target, the olfactory bulb. However, the differential patterns of expression of B-50/GAP43 and OMP at long times after peripheral lesion with TX-100 or bulbectomy illustrate the profound effect the olfactory bulb has on neuronal maturation in reconstituted olfactory neuroepithelium.

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.

Olfactory marker protein (OMP), previously thought to be expressed only by olfactory receptor neurons and their processes, was localized anatomically with immunocytochemical techniques to a number of brain regions in three rodent species, the mouse, rat, and hamster. In addition, the amount of antigen was quantified by radioimmunoassay (RIA) and characterized by an immunoblot procedure. In all three species the antigen could be detected immunocytochemically in the preoptic region and hypothalamus. The rat did not exhibit immunostaining in any other brain region. However, in the mouse neuronal labelling was observed throughout the neural axis, including cellular labelling in the bed nucleus of the anterior commissure, the median preoptic nucleus, the bed nucleus of the stria terminalis, the periventricular region, the anterior parvicellular subnucleus of the paraventricular nucleus, around the dorsomedial hypothalamic nucleus (pars compacta), the subincertal region, the arcuate nucleus, the anterior cortical nucleus of the amygdala, the suprageniculate nucleus, the lateral lemniscal nuclei, the lateraldorsal and lateralventral central gray, the posterior aspects of the commissural and marginal nuclei of the inferior colliculus, the paragenule nucleus, the A-5 region, the area postrema, the ventromedial nucleus of the solitary tract, area X, the spinal trigeminal nucleus (pars zonale), and superficial laminae of the spinal cord. The hamster displayed a different pattern of labelling including cells in the periventricular gray, the pontine reticular tegmental nucleus, the A-5 region, the medial vestibular complex, the prepositus hypoglossal nucleus, the parvicellular reticular nucleus, the lateral paragigantocellular nucleus, the raphe obscuras, the lateral reticular nucleus, and the lateral nucleus of the cerebellum. Immunostaining was seen in fibers within the red nucleus and within mossy fibers of the cerebellum. OMP levels could only be quantified by radioimmunoassay in the olfactory bulb of the three species and in the hamster cerebellum where they were 1/1,000 of those determined in the olfactory bulb. The authenticity of OMP measured in the RIA and detected immunocytochemically was verified by a double-antibody immunoisolation/immunodetection procedure, which confirmed that the antigen being visualized had the molecular properties expected for OMP. In summary, these experiments demonstrate that authentic OMP exists in small groups of neurons in many areas of the central nervous system.