Laminar disorganisation of mitral cells in the olfactory bulb does not affect topographic targeting of primary olfactory axons.

Primary olfactory neurons expressing the same odorant receptor protein typically project to topographically fixed olfactory bulb sites. While cell adhesion molecules and odorant receptors have been implicated in guidance of primary olfactory axons, the postsynaptic mitral cells may also have a role in final target selection. We have examined the effect of disorganisation of the mitral cell soma layer in mutant mice heterozygous for the beta-subunit of platelet activating factor acetylhydrolase (Lis1(-/+)) on the targeting of primary olfactory axons. Lis1(-/+) mice display abnormal lamination of neurons in the olfactory bulb. Lis1(-/+) mice were crossed with the P2-IRES-tau:LacZ line of transgenic mice that selectively expresses beta-galactosidase in primary olfactory neurons expressing the P2 odorant receptor. LacZ histochemistry revealed blue-stained P2 axons that targeted topographically fixed glomeruli in these mice in a manner similar to that observed in the parent P2-IRES-tau:LacZ line. Thus, despite the aberrant organisation of postsynaptic mitral cells in Lis1(-/+) mice, primary olfactory axons continued to converge and form glomeruli at correct sites in the olfactory bulb. Next we examined whether challenging primary olfactory axons in adult Lis(-/+) mice with regeneration would affect their ability to converge and form glomeruli. Following partial chemical ablation of the olfactory neuroepithelium with dichlobenil, primary olfactory neurons die and are replaced by newly differentiating neurons that project axons to the olfactory bulb where they converge and form glomeruli. Despite the aberrant mitral cell layer in Lis(-/+) mice, primary olfactory axons continued to converge and form glomeruli during regeneration. Together these results demonstrate that the convergence of primary olfactory axons during development and regeneration is not affected by gross perturbations to the lamination of the mitral cell layer. Thus, these results support evidence from other studies indicating that mitral cells do not play a major role in the convergence and targeting of primary olfactory axons in the olfactory bulb.

Expression of glycoproteins in the vomeronasal organ reveals a novel spatiotemporal pattern of sensory neurone maturation.

The main olfactory and the accessory olfactory systems are both anatomically and functionally distinct chemosensory systems. The primary sensory neurones of the accessory olfactory system are sequestered in the vomeronasal organ (VNO), where they express pheromone receptors, which are unrelated to the odorant receptors expressed in the principal nasal cavity. We have identified a 240 kDa glycoprotein (VNO(240)) that is selectively expressed by sensory neurones in the VNO but not in the main olfactory neuroepithelium of mouse. VNO(240) is first expressed at embryonic day 20.5 by a small subpopulation of sensory neurones residing within the central region of the crescent-shaped VNO. Although VNO(240) was detected in neuronal perikarya at this age, it was not observed in the axons in the accessory olfactory bulb until postnatal day 3.5. This delayed appearance in the accessory olfactory bulb suggests that VNO(240) is involved in the functional maturation of VNO neurones rather than in axon growth and targeting to the bulb. During the first 2 postnatal weeks, the population of neurones expressing VNO(240) spread peripherally, and by adulthood all primary sensory neurones in the VNO appeared to be expressing this molecule. Similar patterns of expression were also observed for NOC-1, a previously characterized glycoform of the neural cell adhesion molecule NCAM. To date, differential expression of VNO-specific molecules has only been reported along the rostrocaudal axis or at different apical-basal levels in the neuroepithelium. This is the first demonstration of a centroperipheral wave of expression of molecules in the VNO. These results indicate that mechanisms controlling the molecular differentiation of VNO neurones must involve spatial cues organised, not only about orthogonal axes, but also about a centroperipheral axis. Moreover, expression about this centroperipheral axis also involves a temporal component because the subpopulation of neurones expressing VNO(240) and NOC-1 increases during postnatal maturation.

Dynamic spatiotemporal expression patterns of neurocan and phosphacan indicate diverse roles in the developing and adult mouse olfactory system.

The chondroitin sulfate proteoglycans neurocan and phosphacan are believed to modulate neurite outgrowth by binding to cell adhesion molecules, tenascin, and the differentiation factors heparin-binding growth-associated molecule and amphoterin. To assess the role of these chondroitin sulfate proteoglycans in the olfactory system, we describe here their expression patterns during both embryonic and postnatal development in the mouse. Immunoreactivity for neurocan was first detected in primary olfactory neurons at embryonic day 11. 5 (E11.5). Neurocan was expressed by primary olfactory axons as they extended toward the rostral pole of the telencephalon as well as by their arbors in glomeruli after they contacted the olfactory bulb. The role of neurocan was examined by growing olfactory neurons on an extracellular matrix substrate containing neurocan or on extracellular matrix in the presence of soluble neurocan. In both cases, neurocan strongly promoted neurite outgrowth. These results suggest that neurocan supports the growth of primary olfactory axons through the extracellular matrix as they project to the olfactory bulb during development. Phosphacan, unlike neurocan, was present within the mesenchyme surrounding the E11.5 and E12.5 nasal cavity. This expression decreased at E13.5, concomitant with a transient appearance of phosphacan in nerve fascicles. Within the embryonic olfactory bulb, phosphacan was localised to the external and internal plexiform layers. However, during early postnatal development phosphacan was concentrated in the glomerular layer. These results suggest that phosphacan may play a role in delineating the pathway of growing olfactory axons as well as defining the laminar organization of the bulb. Together, the spatiotemporal expression patterns of neurocan and phosphacan indicate that these chondroitin sulfate proteoglycans have diverse in situ roles, which are dependent on context-specific interactions with extracellular and cell adhesion molecules within the developing olfactory nerve pathway.

Expression and analysis of heparin-binding regions of the amyloid precursor protein of Alzheimer's disease.

Deletion mutagenesis studies have suggested that there are two domains within APP which bind heparan sulphate. These domains have been cloned and expressed in the yeast Pichia pastoris. Both recombinant proteins bound to heparin. One domain (APP316-447) was further characterised by binding studies with peptides encompassing this region. Peptides homologous to APP316-346 and APP416-447 were found to bind heparin. Circular dichroism studies show that APP416-447 shifted towards an alpha-helical conformation in the presence of heparin. This study suggests that heparin-binding domains may lie within regions high in alpha-helical structure.

Identification of heparin-binding domains in the amyloid precursor protein of Alzheimer's disease by deletion mutagenesis and peptide mapping.

Recent studies have shown that the binding of the amyloid protein precursor (APP) of Alzheimer's disease to heparan sulfate proteoglycans (HSPGs) can modulate a neurite outgrowth-promoting function associated with APP. We used three different approaches to identify heparin-binding domains in APP. First, as heparin-binding domains are likely to be within highly folded regions of proteins, we analyzed the secondary structure of APP using several predictive algorithms. This analysis showed that two regions of APP695 contain a high degree of secondary structure, and clusters of basic residues, considered mandatory for heparin binding, were found, principally within these regions. To determine which domains of APP bind heparin, deletion mutants of APP695 were prepared and analyzed for binding to a heparin affinity column. The results suggested that there must be at least two distinct heparin-binding regions in APP. To identify novel heparin-binding regions, peptides homologous to candidate heparin-binding domains were analyzed for their ability to bind heparin. These experiments suggested that APP contains at least four heparin-binding domains. The presence of more than one heparin-binding domain on APP suggests the possibility that APP may interact with more than one type of glycosaminoglycan.

Expression of the amyloid protein precursor of Alzheimer's disease in the developing rat olfactory system.

The expression of the amyloid protein precursor (APP) of Alzheimer's disease (AD) was examined in the olfactory system of the developing rat. Two monoclonal antibodies were used to detect APP: Alz-90, which specifically recognizes APP, and 22C11 which recognizes both APP and the structurally related protein APLP-2. Very similar patterns of immunoreactivity were observed with both antibodies. APP immunoreactivity was first detected in a subpopulation of olfactory epithelial cells at embryonic day 16 (E16), at a time when primary sensory olfactory axons are first beginning to pierce the glia limitans of the olfactory bulb. At E16, there were more olfactory receptor neurons which expressed APP than the olfactory marker protein (OMP), indicating that some APP-containing neurons were not fully mature. Between E16 and postnatal day 8 (P8), there was a marked increase in the number of primary sensory olfactory neurons expressing APP. In the olfactory bulb, APP was first detected in the mitral cell layer at E18, at a time when synapses are first beginning to form between the dendrites of these cells and primary sensory axons. The level of APP detected within mitral cell perikarya decreased after birth and could no longer be detected between P3 and P8. This indicated that once synaptic connections had been initiated within olfactory glomeruli, the expression of APP within the mitral cells was down-regulated. High levels of APP were, however, detected within the olfactory nerve fiber layer and glomeruli between P3 and P8. The results demonstrate that APP expression in the olfactory system is coordinately regulated with the major periods of synaptogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of nerve growth factor from septum stimulates neurite outgrowth and release of the amyloid protein precursor of Alzheimer's disease from hippocampal explants.

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular and intracellular compartments of the cerebral cortex. The extracellular amyloid consists of a protein (beta A4) which is derived from a larger precursor, the amyloid protein precursor (APP). Several studies have implicated APP in the regulation of neurite outgrowth during development, although the precise function of APP remains unknown. To examine the role of APP in the regulation of neurite outgrowth from hippocampal neurons, an explant culture system was developed. Explants of E18 mouse hippocampus were found to extend neurites when co-cultured with explants of E18 mouse septum. This finding demonstrated that the septum can release a neurite outgrowth-promoting factor (NOPF). As nerve growth factor (NGF) was also able to stimulate neurite outgrowth from the hippocampal explants, this suggested that the NOPF might be NGF. Immunoprecipitation of NGF from septal conditioned medium using a specific monoclonal antibody (27/21) completely blocked the neurite outgrowth-promoting effect, supporting this conclusion. Concomitant with its ability to stimulate neurite outgrowth, NGF stimulated the release of APP from the hippocampal explants. As previous studies have suggested that the binding of APP to heparan sulfate proteoglycans (HSPGs) in the extracellular matrix might be an important step in the regulation of neurite outgrowth by NGF, we examined the effect of APP on neurite outgrowth from dissociated hippocampal cells cultured on various protein substrates. When cells were cultured on a substrate of APP and HSPG, neurite outgrowth was markedly stimulated. No stimulation of neurite outgrowth was seen when neurons were cultured on substrates of either APP or HSPG alone. The results suggest that secreted forms of APP may be involved in stimulating neurite outgrowth from hippocampal neurons and that interactions between APP and HSPG may be important for a neurite outgrowth-promoting function.