By the end of the first week in culture, hippocampal neurons have established a single axon and several dendrites. These 2 classes of processes differ in their morphology, in their molecular composition, and in their synaptic polarity (Bartlett and Banker, 1984a, b; Caceres et al., 1984). We examined the events during the first week in culture that lead to the establishment of this characteristic form. Hippocampal cells were obtained from 18 d fetal rats, plated onto polylysine- treated coverslips, and maintained in a serum-free medium. The development of individual cells was followed by sequential photography at daily intervals until both axons and dendrites had been established; identification of the processes was confirmed by immunostaining for MAP2, a dendritic marker. Time-lapse video recording was used to follow the early stages of process formation. Hippocampal neurons acquired their characteristic form by a stereotyped sequence of developmental events. The cells first established several, apparently identical, short processes. After several hours, one of the short processes began to grow very rapidly; it became the axon. The remaining processes began to elongate a few days later and grew at a much slower rate. They became the cell's dendrites. Neurons that arose following mitosis in culture underwent this same sequence of developmental events. In a few instances, 2 processes from a cell exhibited the rapid growth typical of axons, but only one maintained this growth; the other retracted and became a dendrite. Axons branched primarily by the formation of collaterals, not by bifurcation of growth cones. As judged by light microscopy, processes are not specified as axons or dendrites when they arise. The first manifestation of neuronal polarity is the acquisition of axonal characteristics by one of the initial processes; subsequently the remaining processes become dendrites.

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called ␤-amyloid (A␤) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APP sec ). It is A␤ that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-␤-cyclodextrin, we show that the formation of A␤ is completely inhibited while the generation of APP sec is unperturbed. This inhibition of A␤ formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for A␤ formation to occur and imply a link between cholesterol, A␤, and Alzheimer's disease.

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The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein A beta. Proteolysis by gamma-secretase is the last processing step resulting in release of A beta. Cleavage occurs after residue 40 of A beta [A beta(1-40)], occasionally after residue 42 [A beta(1-42)]. Even slightly increased amounts of this A beta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD. Unexpectedly we have identified in neurons the endoplasmic reticulum (ER) as the site for generation of A beta(1-42) and the trans-Golgi network (TGN) as the site for A beta(1-40) generation. It is interesting that intracellular generation of A beta seemed to be unique to neurons, because we found that nonneuronal cells produced significant amounts of A beta(1-40) and A beta(1-42) only at the cell surface. The specific production of the critical A beta isoform in the ER of neurons links this compartment with the generation of A beta and explains why primarily ER localized (mutant) proteins such as the presenilins could induce AD. We suggest that the earliest event taking place in AD might be the generation of A beta(1-42) in the ER.

The role of localized instability of the actin network in specifying axonal fate was examined with the use of rat hippocampal neurons in culture. During normal neuronal development, actin dynamics and instability polarized to a single growth cone before axon formation. Consistently, global application of actin-depolymerizing drugs and of the Rho-signaling inactivator toxin B to nonpolarized cells produced neurons with multiple axons. Moreover, disruption of the actin network in one individual growth cone induced its neurite to become the axon. Thus, local instability of the actin network restricted to a single growth cone is a physiological signal specifying neuronal polarization.

The sprouting of neurites, which will later become axons and dendrites, is an important event in early neuronal differentiation. Studies in living neurons indicate that neuritogenesis begins immediately after neuronal commitment, with the activation of membrane receptors by extracellular cues. These receptors activate intracellular cascades that trigger changes in the actin cytoskeleton, which promote the initial breakdown of symmetry. Then, through the regulation of gene transcription, and of microtubule and membrane dynamics, the newly formed neurite becomes stabilized. A key challenge is to define the molecular machinery that regulates the actin cytoskeleton during initial neurite sprouting. We propose that analysing the molecules involved in actin-dependent mechanisms in non-neuronal systems, such as budding yeast and migrating fibroblasts, could help to uncover the secrets of neuritogenesis.

Abstract. Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4-nitrobenzo-2-oxa-l,3-diazole]) aminocaproyl sphingosine.1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved >10 ttm upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Racassociated protein NAP1 and a member of the NSF/ sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.