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For years, extracellular deposition of the “pathologic” β-amyloid1–42 (Aβ42) in the brain has been considered as the major cause of the appearance of neuritic plaques that are subsequently responsible for neuronal death in Alzheimer's disease (AD). However, recent work has shown that significant amounts of β-amyloid, especially Aβ42, are being sequestered within the perikaryon of affected pyramidal neurons prior to the appearance of plaques. This has led us to challenge the prevailing theory and suggest that at least some of the amyloid plaques, specifically the dense-core plaques in the cerebrum and hippocampus, may derive from the lysis of those neurons that are overburdened with intracellular Aβ42. We discuss the possible role of intracellular Aβ42 accumulation in the development of AD pathology and review an alternate hypothesis, where Aβ42 is first accumulated inside the neurons and later released out to the brain parenchyma via neuronal lysis finally forming a dense-core plaque (“Inside-Out” hypothesis). We describe a mechanism for Aβ42 internalization into neurons that involves the high affinity interaction between Aβ42 and the alpha7 nicotinic acetylcholine receptor (α7nAChR) and the internalization and intracellular accumulation of the Aβ42/α7nAChR complex. This alternative hypothesis can account for many of the well-known features of AD pathology, including specific cholinergic and cholinoceptive neuronal and synaptic loss that affects cognitive and memory functions, the distribution, morphology and composition of dense-core plaques, and their association with inflammation. More importantly, it provides the scientific rationale for targeting the mechanisms that lead to the intraneuronal Aβ42 as a novel strategy for AD drug discovery. Drug Dev. Res 56:194–200, 2002. © 2002 Wiley-Liss, Inc.

The origin of the driving forces for neural tube formation remains uncertain but is currently thought to involve the participation of microfilament bundles situated in the apical ends of neuroepithelial cells. In the work presented here, we show how morphometric measurements that map local variations in the apical geometry of neuroepithelial cells (especially apical constriction) can provide information on the distribution of motive forces within the neuroepithelium during neural tube formation. When used in combination with computer-assisted, three-dimensional reconstruction, it becomes possible to analyze the morphometric data from a dynamic, three-dimensional perspective. As an example application of this method, we have used morphometry to evaluate the effects of ionomycin on the developing neuroepithelium. Treatment of early (stages 6-8) chick embryos with 5 microM ionomycin was found to cause rapid bending of the neuroepithelium within 1 min of exposure and a dramatic acceleration of the normal sequence of neural tube formation. Electron microscopy and morphometry revealed that this acceleration was coincident with a marked increase in the local degree of apical constriction of neuroepithelial cells, presumably a consequence of enhanced contractile activity of apical microfilament bundles. This work shows that transient elevation of free calcium levels can accelerate the usual sequential phases of NT formation. The rapidity of the response (hours of normal development reduced to minutes), increased prominence of apical microfilament bundles, and the enhanced degree of apical constriction strongly support a direct causal role for apical microfilament bundles and apical constriction of neuroepithelial cells in bending of the neuroepithelium.

of Alzheimer’s disease (AD), and it is characterized by the deposition of beta-amyloid (A ) in cerebral cortical and meningeal blood vessels (Kawai M. et al., 1993), inducing degeneration of vascular cells. Semicarbazide-sensitive amine oxidase (SSAO) [E.C. 1.4.3.6] is present in vascular cells and in plasma. It metabolizes primary amines (Lyles GA., 1996) generating hydrogen peroxide (H2O2), ammonia (NH3) and the corresponding aldehyde, that contribute to the oxidative stress, advanced glycation end-product generation (GubisneHaberle D. et al., 2004), and beta amyloid aggregation (Chen K. et al., 2006). Furthermore in endothelial cells, SSAO is induced under inflammatory conditions (Smith D.J. et. al., 1998). We have reported that SSAO is overexpressed in cerebrovascular tissue of patients with CAA-AD, and that it colocalizes with beta-amyloid deposits (Ferrer I. et al., 2002). This over-expression correlates with high SSAO activity in plasma of severe AD patients (del Mar Hernandez M. et al., 2005). We have also described that plasma SSAO is able to induce apoptosis in vascular cells (Hernandez M. et.al., 2006). The aim of this work is to demonstrate whether A is able to induce SSAO overexpression in HUVEC cells as vascular cell type. Methods: Because of the SSAO/ VAP-1 expression fenotype is lost in cultured cells, HUVEC (human umbilical vein endothelial cells) cells, were stably transfected with vector pcDNA 3.1 containing hVAP-1/SSAO. Cells were treated with A 1-40 Dutch type (mutation E22Q) and/or Methylamine as SSAO substrate. Cell viability, SSAO activity and its expression were determined using specific antibodies against SSAO. Results: Herein we report that A 1-40 E22Q induces the SSAO activity and expression in HUVEC cells. This increasing activity promotes oxidative stress that enhances the toxicity generated by A alone. This toxicity is reverted by specific SSAO inhibitors, confirming SSAO as the responsible of such effect. Conclusions: These results allow us to postulate that deposits of A in cerebrovascular tissue, induce SSAO expression that may contribute to the vascular damage associated to CAA-AD.

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.

The effects of diazepam on the development of explanted stage 4 chick embryos were investigated. Diazepam, at 10-120 micrograms/ml, preferentially inhibited closure of the neural tube. This effect was reversible. Concentrations of 150-200 micrograms/ml inhibited not only neural tube closure but also blastodermal expansion, somite formation, and heart development in 52% of the embryos. Concentrations above 200 micrograms/ml were highly embryotoxic. Electron microscopy of affected neuroepithelial cells revealed that 1) apical surfaces were much smoother than usual and 2) apical filament bundles, which are generally thought to provide motive forces for uplifting of neural folds, were not well organized and often lacked alternating dark and light areas along their length. These findings and the fact that changes in cell surface topography reflect the contractile activities of underlying filament bundles suggest that the observed "smoothing" effect on apical cell surfaces and neural tube closure defects are due, at least in part, to the impaired ability of these filament bundles to contract.

The toxic and teratologic effects of caffeine on chick embryos explanted at stages 4-7 and cultured for 19-22 hours were investigated. Caffeine, at 200-300 micrograms/ml, significantly increased the incidence of neural tube defects regardless of the developmental stage at treatment. Concentrations of 500 micrograms/ml or higher inhibited morphogenesis of nearly all organ primordia. In general, the effects of caffeine were concentration dependent and younger embryos were more susceptible to treatment than their older counterparts. Microscopic studies confirmed that the developing neuroepithelium was most sensitive to treatment. Caffeine, at concentrations sufficient to inhibit neural tube closure, caused no apparent alteration in the ultrastructure of cellular components except that apical microfilament bundles were thinner and less conspicuous than usual. Furthermore, caffeine (400 micrograms/ml) selectively inhibited uplifting of neural folds (and hence, closure of the neural tube) in embryos explanted at stage 8 and cultured for 4-6 and 16 hours. Affected neuroepithelial cells lacked the typical bottle-shaped characteristic and folded apical surfaces. Overall results of this study suggest that caffeine causes neural tube defects, at least in part, through its inhibitory action on the contractile activity of apical microfilament bundles in developing neuroepithelial cells.

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197–205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of developing neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).

Primordial germ cells (PGCs) of the early chick embryo were examined using scanning electron microscopy. Temporal changes in the form and distribution of surface projections were found to be correlated with migratory phases of PGCs. Non-migrating PGCs were spherical to ovoid with relatively smooth surfaces. Their transition to the migratory phase was first evidenced by a burst of membrane activity. Migrating PGCs became somewhat flattened against the underlying hypoblast (which serves as the substratum for their migration) and exhibited blebs and lamellar processes. The lamellar processes were most prominent at the leading edges of actively migrating PGCs. Overall results of the present study indicates that PGCs found in the germinal crescent area of early chick embryos actively migrate on the dorsal surface of the hypoblast towards posterior embryonic regions.

The organization and properties of microfilaments of developing chick neuroepithelial cells were investigated in an attempt to elucidate the structural basis for the observed changes in cell shape during uplifting of neural folds. Microfilaments in the apical regions of cells are more organized than those along the lateral and basal cell margins and are associated with developing junctions. At the level of the zonula occludens, microfilaments are arranged into a loose meshwork extending just below the luminal surface. At the level of the zonula adherens, microfilaments are organized into several discrete bundles rather than as a single circumferential bundle. The opposite ends of these bundles appear to make contact with the plasma membrane at specialized areas of the junction. Microfilaments in the peripheral cytoplasm of the neck and basal regions form a continuous but sparse meshwork. All of these microfilaments bind heavy meromyosin. The implications of the present findings to the observed changes in cell shape during uplifting of neural folds in the chick is discussed.

Changes in cell surface morphology of the neuroepithelium during fusion of neural folds in the chick were studied. As the folds were about to meet, a thick extracellular coat material (ECM) appeared between the two leading edges. Cell membranes forming the fusion area were relatively smooth and heavily coated with ECM. By contrast, the apical surface of most cells lining the wall of the neural tube was folded with much less ECM. During the contact of neural folds, ECM was displaced from the space between the two leading edges, leaving a thin, closely adherent "typical" cell surface coat. Trypsin and concanavalin A inhibited proper alignment and fusion of apposing neural folds by modifying the surface of developing neuroepithelium. Results of this study support a hypothesis that ECM may serve temporarily as an adhesive to bind together the leading edges of neural folds until establishment of more intimate contacts (junctional complexes).

Sequential changes in the morphology of early chick embryos were, for the first time, photographically recorded. Embryos were explanted at stage 8 (four-somite) or 9- (six-somite) of development using New's technique and grown in nutrient medium (thin albumen) with or without a teratologic dose (200 micrograms/ml) of xylocaine. They were photographed using a Nikon Diaphot inverted microscope equipped with both phase-contrast optics and photomicrographic accessories maintained in an incubator. It was found, among other things, that a characteristic neural tube closure defect often seen in the midbrain and anterior portion of the hindbrain of xylocaine (200 micrograms/ml)-treated chick embryos was a consequence of failure of the neural tube to withstand the tension generated by the rapidly expanding cephalic region, which occurred, regardless of the stage at explanation, when corresponding control embryos had advanced to stage 10+ (11-somite) of development.

The possible involvement of myosin in elevation of neural folds in the chick was studied. Indirect immunofluorescence revealed the presence of myosin in the neuroepithelium as early as the neural-plate stage and was concentrated in the apical regions of neuroepithelial cells where microfilaments are known to be organized into discrete bundles. This fluorescent pattern persisted until closure of the neural tube. Actin-specific fluorescence followed a similar distribution pattern as myosin. Diazepam (Valium/Roche), at 400 micrograms/ml, was found to preferentially inhibit elevation of neural folds in explanted stage 8 embryos within 6 hr of incubation. Affected neuroepithelial cells were often less elongated, contained thinner and less conspicuous microfilament bundles, and had apical surfaces which were smoother and broader than the controls. These morphological changes were accompanied by a considerable reduction in the intensity of myosin-specific fluorescence, particularly in the cell apices. Results suggest that (1) diazepam inhibits elevation of neural folds through its disruptive effects on the organization and contractility of apical microfilament bundles in developing neuroepithelial cells and (2) myosin may be directly involved in elevation of neural folds.

The biomechanical basis of diazepam (Valium/Roche)-induced neural tube defects in the chick was investigated using a combination of electron microscopy and morphometry. Embryos at stage 8 (four-somite stage) of development were explanted and grown for 6 hr in nutrient medium containing 400 micrograms/ml diazepam. Nearly 80% of these embryos exhibited neural tube defects that were most pronounced in the forming midbrain region and typified by a "relaxation" or "collapse" of neural folds. The hindbrain and spinal cord regions were less affected. Electron microscopy revealed that neuroepithelial cells in diazepam-treated embryos had smoother apical surfaces and broader apical widths than did controls. Morphometric measurements supported this observation and further showed that these effects were focused at sites within the wall of the forming neural tube that typically exhibit the greatest degree of bending and apical constriction (i.e., the floor and midlateral walls). Overall results indicate that neural tube defects associated with exposure to diazepam are due largely to a general inhibition of the contractile activity of apical microfilament bundles in neuroepithelial cells. These findings 1) emphasize the important contribution of microfilament-mediated apical constriction of neuroepithelial cells in providing the driving forces for bending of the neuroepithelium during neural tube formation and 2) suggest that agents or conditions that impair their contractile activity could play a role in the pathogenesis of certain types of neural tube defects.

The distribution of extracellular material (ECM) in developing neuroepithelium of stage 8+ chick embryos was investigated using lanthanum nitrate. The temporal changes in the distribution of ECM was correlated with the ongoing morphogenetic movements. The most striking finding was that as the folds were about to meet, thick dense ECM appeared between leading edges. Concanavalin A inhibited neural tube closure by binding to cell surfaces and disrupting the usual distribution of ECM in developing neuroepithelium.

Toxic and teratologic effects of verapamil, a calcium antagonist, on chick embryos explanted at stage 8 (four-somite stage) and cultured for 6-8 hours were investigated. In general, embryos responded to verapamil in a dose-related manner. Concentrations lower than 2 micrograms/ml had no apparent effect on the development of embryos. A concentration of 15 micrograms/ml significantly increased the incidence of embryos (approximately 80% of viable embryos) with neural tube closure defects and less numerous somites. Higher concentrations (e.g., 30 micrograms/ml) were embryotoxic and over 90% of the embryos were either severely malformed or dead after 8 hours of incubation. Compared to controls, verapamil-treated neuroepithelial cells had smoother apical surfaces and less conspicuous microfilament bundles. The deleterious effects of verapamil (15 micrograms/ml) could be reversed by subculturing the affected embryos, within 3 hours of treatment, on nutrient medium alone or on nutrient medium containing 25 micrograms/ml chlorotetracycline (CTC), a calcium agonist, the latter being more effective provided that treatment did not exceed 4 hours. Exposure of the developing neuroepithelium to 15 micrograms/ml verapamil for 3-4 hours resulted in a significant reduction in free Ca2+ levels, as revealed by the pyroantimonate precipitation method, throughout neuroepithelial cells. Overall results suggest that verapamil causes neural tube closure defects by reducing intracellular free Ca2+ levels, thereby relaxing apical microfilament bundles of developing neuroepithelial cells.