Neurine, an acetylcholine autolysis product, elevates secreted amyloid-beta protein precursor and amyloid-beta peptide levels, and lowers neuronal cell viability in culture: a role in Alzheimer's disease?

Classical hallmarks of Alzheimer's disease (AD) are a synaptic loss, cholinergic neuron death, and abnormal protein deposition, particularly of toxic amyloid-beta peptide (Abeta) that is derived from amyloid-beta protein precursor (AbetaPP) by the action of beta- and gamma-secretases. The trigger(s) initiating the biochemical cascades that underpin these hallmarks have yet to be fully elucidated. The typical forebrain cholinergic cell demise associated with AD brain results in a loss of presynaptic cholinergic markers and acetylcholine (ACh). Neurine (vinyl-trimethyl-ammonium hydroxide) is a breakdown product of ACh, consequent to autolysis and is an organic poison found in cadavre brain. The time- and concentration-dependent actions of neurine were assessed in human neuroblastoma (NB, SK-N-SH) cells in culture by quantifying cell viability by lactate dehydrogenase (LDH) and MTS assay, and AbetaPP and Abeta levels by Western blot and ELISA. NB cells displayed evidence of toxicity to neurine at > or = 3 mg/ml, as demonstrated by elevated LDH levels in the culture media and a reduced cell viability shown by the MTS assay. Using subtoxic concentrations of neurine, elevations in AbetaPP and Abeta1-40 peptide levels were detected in conditioned media samples.

Immunohistochemistry of the canine vomeronasal organ.

The canine's olfactory acuity is legendary, but neither its main olfactory system nor its vomeronasal system has been described in much detail. We used immunohistochemistry on paraffin-embedded sections of male and female adult dog vomeronasal organ (VNO) to characterize the expression of proteins known to be expressed in the VNO of several other mammals. Basal cell bodies were more apparent in each section than in rodent VNO and expressed immunoreactivity to anticytokeratin and antiepidermal growth factor receptor antibodies. The thin layer of neurone cell bodies in the sensory epithelium and axon fascicles in the lamina propria expressed immunoreactivity to neurone cell adhesion molecule, neurone-specific beta tubulin and protein gene product 9.5. Some neurones expressed growth-associated protein 43 (GAP43): and a number of those also expressed neurone-specific beta tubulin-immunoreactivity. Some axon fascicles were double labelled for those two proteins. The G-protein alpha subunits Gi and Go, involved in the signal transduction pathway, showed immunoreactivity in the sensory cell layer. Our results demonstrate that the canine vomeronasal organ contains a population of cells that expresses several neuronal markers. Furthermore, GAP43 immunoreactivity suggests that the sensory epithelium is neurogenic in adult dogs.

Ultrastructural analysis of the paranodal junction of myelinated fibers in 31-month-old-rats.

Recent studies have revealed a significant decrease in white matter volume, including loss of myelin, with age but minimal decrease in gray matter volume (Guttmann et al., [1998] Neurology 50:972-978). Myelin is necessary for the rapid conduction of impulses along axons. Myelinated nerve includes various domains, the node of Ranvier, the paranodal region, the juxtaparanodal region and the internode. The paranodal junction may serve to anchor the myelin sheath to the axon. We analyzed the ultrastructure of the paranodal region in myelinated fibers from the aged rat brain. Severe alterations of myelinated fibers were observed in 31-month-old rats, resulting in the appearance of macrophages, splitting of the myelin sheath, myelin balloon formation and separation from the axon. Many paranodal retractions of myelinated axons occurred in the aged rats. It should be noted that the paranodal junction is functionally important, serving to anchor the myelin to the axon and that there is a diffusion barrier in the paranodal region. We analyzed myelin-related proteins from young and aged rat brains. The 21.5-kDa isoform of myelin basic protein (MBP) almost disappeared in the 31-month-old rats, whereas other myelin proteins were not significantly changed between young and aged rats. These results suggest that this isoform, a highly cationic charged major dense component protein that binds lipid bilayer in the membrane, may participate in the formation of a paranodal diffusion barrier at the myelin/noncompact membrane border.

The cell adhesion molecule BEN defines a prosensory patch in the developing avian otocyst.

The distribution of the cell adhesion molecule BEN in the developing chick inner ear is described. BEN is first detected in the otic placode at stage 11. As the placode begins to invaginate, BEN becomes concentrated in a ventromedial region extending from the anterior to the posterior end of the otic pit. BEN expression levels increase in this region as the pit closes to form the otocyst, and distinct boundaries become defined along the dorsal and ventral edges of the ventromedial band of BEN expression. BEN expression also becomes concentrated dorsally within the otic epithelium as the pit closes and is observed in the condensing otic ganglion. By stage 22, the ventromedial band of BEN expression splits into two distinct regions, a small caudal patch within which the posterior crista will develop, and a larger anterior patch. By stage 26, this larger anterior patch of cells expressing BEN becomes subdivided into five separate areas corresponding to the regions within which the anterior crista, the lateral crista, the utricle, the saccule, and both the basilar papilla and lagenar macula form. Hair cells only develop within these regions defined by BEN distribution. The data suggest that the ventromedial patch of BEN expression observed from stage 11 onwards defines a single sensory competent zone from which all sensory organs of the inner ear develop. BEN immunoreactivity in the inner ear declines after stage 38. In response to noise exposure, upregulation of BEN expression is mainly detected in regions of the posthatch papilla where the damage is severe and regenerating hair cells are not observed. The regenerating hair and supporting cells do not express BEN, highlighting a molecular difference between the processes of development and regeneration.

Clustering and functional cooperation of Ng-CAM and axonin-1 in the substratum-contact area of growth cones.

Growth cones and neurites of chicken dorsal root ganglia neurons cultured on laminin, Ng-CAM, or axonin-1 exhibit substratum-dependent morphology and growth patterns which are accompanied by distinctive distributions of axonin-1 and Ng-CAM in the growth cone membrane. On either Ng-CAM or axonin-1 substratum, both Ng-CAM and axonin-1 were depleted from some areas of the apical growth cone membrane. In contrast, on laminin, both axonin-1 and Ng-CAM remained randomly distributed. Removal of axonin-1 from growth cones resulted in a blockage of neurite outgrowth on both Ng-CAM and axonin-1 substrata, indicating that in these neurons axonin-1 cooperates with Ng-CAM in the activation of axon growth. Based on these results possible molecular models for cooperation between axonin-1 and Ng-CAM on the growth cone are discussed.

Expression of the axonal cell adhesion molecules axonin-1 and Ng-CAM during the development of the chick retinotectal system.

Cell surface glycoproteins expressed on growth cones and axons during brain development have been postulated to be involved in the cell-cell interactions that guide axons into their target area. Nevertheless, an unequivocal description of the mechanism by which such molecules exert control over the pathway of a growing axon has not been done. As a crucial requirement in support of a relevant involvement of an axonal surface molecule in growth cone guidance, this molecule should be expressed in the growth cone. The developing retinotectal system provides an excellent opportunity to test whether a particular neuronal surface molecule fulfills the requirement of the spatiotemporal coincidence between its appearance and the emergence of growth cones because its setup follows the rule of chronotopy, i.e., the position of axons in a certain site is determined by the time of their arrival. We have analyzed axonin-1 and the neuron-glia cell adhesion molecule (Ng-CAM), two axonal surface molecules that promote neurite growth in vitro, for their expression in the retina and in the retinotectal system of the chick throughout its development. At stage 18, both axonin-like (A-LI) and Ng-CAM-like immunoreactivity (Ng-CAM-LI) are clearly present in the area where first retinal ganglion cells (RGCs) are generated. The immunoreactivity spreads synchronously with the formation of RGCs over the developing retina. From stage 32 on, the inner plexiform layer is also stained according to its temporospatial gradient of maturation. In later stages, the outer plexiform layer and the inner segments of photoreceptors also show immunoreactivity. The development of A-LI and Ng-CAM-LI along the optic nerve, chiasm, optic tract, and in the superficial layers of the optic tectum follows the chronotopic pattern of axons, as was found by earlier morphological investigations. Older axons loose their A-LI. This allows to localize the position of newly formed axons. The fact that A-LI and Ng-CAM-LI parallel the formation and maturation of axons suggests that axonin-1 and Ng-CAM may play an important role in the organization of the retinotectal system.