Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides.

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.

Routine obstetrical interventions: research agenda for the next decade.

There are many interventions performed as a part of the active management of labor and birth that have remained unchanged over the years. Separating ritual from beneficial nursing care can guide the development of a research agenda. The agenda for the next 10 years can address some of the gaps remaining in the evidence-based foundation for routine interventions used during labor and birth. The gaps identify areas where unanswered questions prevent optimal care from occurring. These unanswered questions include optimal time for epidural administration; management of maternal movement during labor when routine interventions make this difficult; changing the practice of immediate, closed glottis coached pushing at 10 cm; continuous electronic fetal monitoring; routine administration of intravenous fluids; and finally, the use of oxytocin as augmentation or induction of labor.

The XLMR gene ACSL4 plays a role in dendritic spine architecture.

ACSL4 is a gene involved in non-syndromic X-linked mental retardation. It encodes for a ubiquitous protein that adds coenzyme A to long-chain fatty acids, with a high substrate preference for arachidonic acid. It presents also a brain-specific isoform deriving from an alternative splicing and containing 41 additional N-terminal amino acids. To start to unravelling the link between ACSL4 and mental retardation, we have performed molecular and cell biological studies. By retro-transcription polymerase chain reaction analyses we identified a new transcript with a shorter 5'-UTR region. By immunofluorescence microscopy in embryonic rat hippocampal neurons we report that ACSL4 is associated preferentially to endoplasmic reticulum tubules. ACSL4 knockdown by siRNAs in hippocampal neurons indicated that this protein is largely dispensable for these cells' gross architectural features (i.e. axonal and dendritic formation and final length) yet it is required for the presence of normal spines. In fact, reduced levels of ACSL4 led to a significant reduction in dendritic spine density and an alteration in spine/filopodia distribution. The possible mechanisms behind this phenotype are discussed.

Axonal and dendritic endocytic pathways in cultured neurons.

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.

pH-induced microtubule-dependent redistribution of late endosomes in neuronal and epithelial cells.

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to approximately 6.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell.

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium.

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-1,3-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 greater than 10 microns 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.

Peripherin expression in hippocampal neurons induced by muscle soluble factor(s).

Previous studies have shown that neuronal cells in culture can switch neurotransmitters when grown in the presence of different target cells. To examine whether this plasticity extends to structural proteins, we cocultured hippocampal neurons and pituitary-derived neuroendocrine (AtT20) cells with astrocytes, kidney epithelial cells, or skeletal muscle cells. As a marker of phenotypic change we used the cytoskeletal protein peripherin, a type III intermediate filament (IF) subunit which is not expressed in hippocampal neurons and AtT20 cells. We show here that soluble factor(s) secreted specifically from skeletal muscle cells can induce the expression and de novo assembly of peripherin in a subset of post-mitotic neurons. We further demonstrate that one of these factors is the Leukemia Inhibitory Factor/Cholinergic Neuronal Differentiation Factor. The environmentally regulated expression of peripherin implies a remarkable degree of plasticity in the cytoskeletal organization of postmitotic CNS cells and provides a noninvasive model system to examine the de novo assembly of IF proteins under in vivo conditions.

Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture.

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.

Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p.

In the companion paper (Huber, L. A., S. W. Pimplikar, R. G. Parton, H. Virta, M. Zerial, and K. Simons. J. Cell Biol. 123:35-45) we reported that the small GTPase rab8p is involved in transport from the TGN to the basolateral plasma membrane in epithelia. In the present work we investigated the localization and function of rab8p in polarized hippocampal neurons. By immunofluorescence microscopy we found that rab8p localized preferentially in the somatodendritic domain, and was excluded from the axon. Double-labeling immunofluorescence showed that some of the rab8p co-localized in the dendrites with the Semliki Forest Virus glycoprotein E2 (SFV-E2). An antisense oligonucleotide approach was used to investigate the role of rab8p in dendritic transport of newly synthesized viral glycoproteins. Antisense oligonucleotides corresponding to the initiation region of the rab8 coding sequence were added to the cultured neurons for four days. This treatment resulted in a significant decrease in cellular levels of rab8p and transport of SFV-E2 from the cell body to the dendrites was significantly reduced. However, no effect was observed on axonal transport of influenza HA. From these results we conclude that rab8p is involved in transport of proteins to the dendritic surface in neurons.

The role of local actin instability in axon formation.

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.

An evidence-based consensus statement on the role and application of endosonography in clinical practice.

BACKGROUND: Endoscopic ultrasonography (EUS) has evolved over the past 20 years with the emergence of novel diagnostic and therapeutic indications. Our goal was to identify the best evidence supporting the use of EUS. MATERIALS AND METHODS: A six-step approach was employed to develop recommendations using accepted methodology. Twenty-two experienced endosonographers identified topics and reviewed studies using MeSH (medical subject headings) terminology and free text in PubMed. Medline and society abstracts were reviewed if necessary. The quality of evidence, the strength of recommendations, and level of consensus were graded and voted on. RESULTS: Consensus was reached for several clinical scenarios for which the impact of EUS findings was supported by a high level of evidence. These included diagnosis and staging of esophageal cancer, differential diagnosis of subepithelial lesions, thickened gastric folds, assessment of peritoneal involvement in patients with gastric cancer, mucosa-associated lymphoid tissue lymphoma, diagnosis of common bile duct/gallbladder stones, diagnosis of chronic pancreatitis, differential diagnosis of a solid mass in patients with chronic pancreatitis, differential diagnosis of pancreatic cyst, rectal cancer staging, and diagnosis and staging of non-small-cell lung cancer. The recommendations were adopted by the Brazilian Society of Gastrointestinal Endoscopy. Several indications continue to emerge and require additional validation.

Neuronal polarity: vectorial cytoplasmic flow precedes axon formation.

Axon formation in multipolar neurons is believed to depend on the existence of precise sorting mechanisms for axonal membrane and membrane-associated proteins. Conclusive evidence in living neurons, however, is lacking. In the present study, we use light and video microscopy to address this issue directly. We show that axon formation is preceded by the appearance in one of the multiple neurites of (1) a larger growth cone, (2) a higher amount and greater transport of membrane organelles, (3) polarized delivery of TGN-derived vesicles, (4) a higher concentration of mitochondria and peroxisomes, (5) a higher concentration of a cytosolic protein, and (6) a higher concentration of ribosomes. These results provide evidence for the involvement of bulk cytoplasmic flow as an early determinant of neuronal morphological polarization. Molecular sorting events would later trigger the establishment of functional polarity.

The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis.

Rab5a is a small GTPase that regulates fusion of endocytic vesicles to early endosomes. We investigated whether Rab5a is involved in early endocytic traffic in both the axonal and the somatodendritic domains of polarized neurons. Using immunofluorescence, endogenous Rab5a was detected in axons and dendrites. Its localization in axons strongly overlapped that of the synaptic vesicle protein synaptophysin. Indeed, Rab5a co-immunoisolated with synaptophysin-containing vesicles, and antibodies against Rab5a labeled synaptic vesicle-like structures in nerve terminals. The functional association of Rab5a with dendritic and axonal early endosomes was assayed by electron microscopy after overexpression of wild-type and mutant Rab5a in cultured hippocampal neurons. This induced the formation of abnormal endosomes in both the somatodendritic and the axonal domains. These results show a role for Rab5a in axonal and dendritic endocytosis, and the presence of Rab5a on synaptic vesicles indicates that the axonal endosomes participate in the biogenesis of these vesicles.

Effects of oxytocin-induced uterine hyperstimulation during labor on fetal oxygen status and fetal heart rate patterns.

OBJECTIVE: The objective of the study was to evaluate effects of oxytocin-induced hyperstimulation on fetal oxygen saturation and fetal heart rate patterns. STUDY DESIGN: Uterine activity of 56 women was evaluated retrospectively for hyperstimulation lasting 30 minutes using 2 definitions: group 1: 5 or more but less than 6 contractions in 10 minutes (n = 102, 30-minute periods); group 2: 6 or more contractions in 10 minutes (n = 56, 30-minute periods). Fetal oxygen saturation and heart rate patterns during each period and the preceding 30 minutes of less than 5 contractions in 10 minutes were compared. RESULTS: Hyperstimulation was associated with significant oxygen desaturation: (group 1 = 10.68 [20%] decrease from 52.14 to 41.46; P < .001); group 2 = 15.34 [29%] decrease from 52.02 to 36.68: P < .001) and significantly more nonreassuring fetal heart rate characteristics, compared with normal uterine activity. CONCLUSION: Hyperstimulation is associated with negative effects on fetal status. The more contractions in 30 minutes, the more pronounced the effect.

Differentiated neurons retain the capacity to generate axons from dendrites.

Cutting the axon of a morphologically polarized neuron (stage 3) close to the cell body causes another neurite to grow as an axon [1-3]. Stage 3 neurons still lack molecular segregation of axonal and dendritic proteins, however. Axonal and dendritic compartments acquire their distinct composition at stage 4 (4-5days in culture), when proteins such as the microtubule-associated protein 2 (MAP-2) and the glutamate receptor subunit GluR1 localize to the dendrites and disappear from the axon [4,5]. We investigated whether cultured hippocampal neurons retained axon/dendrite plasticity after axons and dendrites have created their distinct cytoskeletal architecture and acquired their specific membrane composition. We found that axotomy of stage 4 neurons transformed a dendrite into an axon. Using axonal and dendritic markers, we tested whether cytoskeletal changes could cause similar transformations, and found that actin depolymerization induced multiple axons in unpolarized neurons. Moreover, depletion of actin filaments from both morphologically and molecularly polarized cells also resulted in the growth of multiple axons from pre-existing dendrites. These results imply that dendrites retain the potential to become axons even after molecular segregation has occurred and that the dendritic fate depends on the integrity of the actin cytoskeleton.

Transcytosis of the polymeric immunoglobulin receptor in cultured hippocampal neurons.

BACKGROUND: A wide variety of proteins are transported across epithelial cells by vesicular carriers. This process, transcytosis, is used to generate cell surface polarity and to transport macromolecules between the luminal and serosal sides of the epithelial layer. The polymeric immunoglobulin receptor is a well-characterized transcytotic molecule in epithelia. It binds to its ligand, polymeric immunoglobulin, at the basolateral surface, and the receptor-ligand complex is transcytosed to the apical surface, where the ligand is released. Our previous studies have shown that hippocampal neurons may employ mechanisms similar to those of epithelial cells to sort proteins to two plasma membrane domains. The machinery used for axonal delivery recognizes proteins that are targeted apically in epithelia, whereas basolaterally destined proteins are delivered to the dendrites. It has not been clear, however, whether transcytosis occurs in neurons. RESULTS: We report expression of the polymeric immunoglobulin receptor in cultured hippocampal neurons, using a Semliki Forest Virus expression system, and show by immunofluorescence microscopy that the newly synthesized receptor is targeted from the Golgi complex predominantly to the dendrites - only about 20% of the infected neurons display axonal immunofluorescence. Addition of ligand leads to significant redistribution of the receptor to the axons, shown by an approximately three-fold increase in axonal immunoreactivity with the anti-receptor antibodies. CONCLUSIONS: Our results suggest that a transcytotic route, analogous to that in epithelia, exists in neurons, where it transports proteins from the somatodendritic to the axonal domain. Cultured neurons expressing the polymeric immunoglobulin receptor offer an experimental system that should be useful for further characterization of this novel neuronal pathway at the molecular and functional level.

NXF5, a novel member of the nuclear RNA export factor family, is lost in a male patient with a syndromic form of mental retardation.

BACKGROUND: Although X-linked mental retardation (XLMR) affects 2%-3% of the human population, little is known about the underlying molecular mechanisms. Recent interest in this topic led to the identification of several genes for which mutations result in the disturbance of cognitive development. RESULTS: We identified a novel gene that is interrupted by an inv(X)(p21.1;q22) in a male patient with a syndromic form of mental retardation. Molecular analysis of both breakpoint regions did not reveal an interrupted gene on Xp, but identified a novel nuclear RNA export factor (NXF) gene cluster, Xcen-NXF5-NXF2-NXF4-NXF3-Xqter, in which NXF5 is split by the breakpoint, leading to its functional nullisomy. The predicted NXF5 protein shows high similarity with the central part of the presumed mRNA nuclear export factor TAP/NXF1. Functional analysis of NXF5 demonstrates binding to RNA as well as to the RNA nuclear export-associated protein p15/NXT. In contrast to TAP/NXF1, overexpression studies localized NXF5 in the form of granules in the cell body and neurites of mature hippocampal neurons, suggesting a role in mRNA transport. The two newly identified mouse nxf homologs, nxf-a and nxf-b, which also map on X, show highest mRNA levels in the brain. CONCLUSIONS: A novel member of the nuclear RNA export factor family is absent in a male patient with a syndromic form of mental retardation. Although we did not find direct evidence for the involvement of NXF5 in MR, the gene could be involved in development, possibly through a process in mRNA metabolism in neurons.

A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons.

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.

A human sequence homologue of Staufen is an RNA-binding protein that is associated with polysomes and localizes to the rough endoplasmic reticulum.

In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a lambda cDNA library. The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparent Kd of 10(-9) M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes. These results are discussed in relation to the possible functions of the protein.

Spreading in narrow channels.

We study a lattice model for the spreading of fluid films, which are a few molecular layers thick, in narrow channels with inert lateral walls. We focus on systems connected to two particle reservoirs at different chemical potentials, considering an attractive substrate potential at the bottom, confining sidewalls, and hard-core repulsive fluid-fluid interactions. Using kinetic Monte Carlo simulations we find a diffusive behavior. The corresponding diffusion coefficient depends on the density and is bounded from below by the free one-dimensional diffusion coefficient, valid for an inert bottom wall. These numerical results are rationalized within the corresponding continuum limit.