Toxicity of tunicamycin to cultured brain neurons: ultrastructure of the degenerating neurons.

Our previous study has shown that tunicamycin irreversibly downregulates the expression of GABA(A)R and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mechanisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brains were incubated with 5 microg/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons treated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), nuclear envelope and components of Golgi apparatus, in addition to the degranulation of rER and disaggregation of ribosomal rosettes. In neurons subjected to the prolonged incubation, some ribosomes reattached to the membranes of rER; the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle-like structure appeared in the perikarya. This structure is implicated to involve the neuronal degeneration. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons resemble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA-elicited current in the neurons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM treatment to the neurons and suggest differential mechanisms for the neuronal death among various populations of cells. It is evident that the N-glycosylation plays a critical role for neuronal survival.     

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.     

Signaling on the endocytic pathway

Endocytosis regulates many cellular signaling processes by controlling the number of functional receptors available at the cell surface. Conversely, some signaling processes regulate the endocytic pathway. Furthermore, various cellular signaling events appear to occur on endosome membranes. The endocytic pathway, by providing a set of dynamic and biochemically specialized endomembrane structures that physically communicate with the plasma membrane, is increasingly viewed as a highly flexible scaffold for mediating precise spatiotemporal control and transport of diverse biological signals. General principles of endosome-based signaling are beginning to emerge but, in many cases, the physiological significance of signaling on the endocytic pathway remains poorly understood.     

Phosphoinositides and the endocytic pathway

Phosphorylation of the phosphatidylinositol headgroup generates seven varieties of phosphoinositide of which PtdIns(4,5)P₂, PtdIns3P and PtdIns (3,5)P₂ have established roles on the endocytic pathway. In this review, we discuss the enzymes responsible for generation and turnover of these lipids, which are keys to determining compartmental identity and the flux of material through the endocytic system. The enzymatic generation of lipids serves as an amplification mechanism through which a wide variety of effector molecules can be recruited.     

The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells

The structure of cellular organelles, in particular those involved in endocytosis, was studied by electron microscopy with hydrated cryosections. In this technique no chemical treatment is used, and the native structure of organelles can be observed in sections viewed at temperatures below -140 degrees C, using a cold stage accessory on the electron microscope. The compartments of the endocytic pathway were prelabeled with gold markers in the living cell, facilitating the identification of different structures in the cryosections. The structure of most identifiable cellular organelles, including those involved in endocytosis, appeared very similar in the hydrated cryosections to that seen after conventional plastic and cryosections of chemically fixed cells. In particular, the internal membranes of the structure we refer to as the prelysosomal compartment (Griffiths et al., Cell 52, 329-341 (1988] could be clearly visualized in these sections indicating that the organization of these membranes is not a consequence of the chemical fixation process.     

ESCRTing proteins in the endocytic pathway

The endosomal sorting complex required for transport (ESCRT) machinery is highly conserved and its components have been found in all five major supergroups of eukaryotes. The three ESCRT complexes and associated proteins play critical roles in receptor downregulation, retroviral budding, and other normal and pathological cellular processes. Besides monoubiquitin-dependent protein cargo recognition and sorting, the ESCRT machinery also appears to drive the formation of multivesicular bodies (MVBs). Recent advances in the determination of the function and structure of the ESCRT complexes have improved our understanding of the molecular details underlying the assembly and regulation of the ESCRT machinery.     

Effect of theophylline on the glomerular pathway of ferritin macromolecules in rats]

The present microscopic study evaluates the quantitative variations of ferritin particules density within the glomerular ultrastructures after theophyllin perfusion in the Rat. The ferritin particules density increases 3,65 times in the glomerular basement membrane and decreases 3,28 times in the glomerular capillary lumen. So, theophyllin increases considerably the glomerular pathway of the protein macromolecules. This results confirm the increased volumic flow (Jv) and macro-solutes flow (Js') noted with clearance methods and urinary excretion studies.     

Effect of monensin on cell ultrastructure and glycoprotein migration in adult mouse jejunal epithelium in organ culture

Summary Expiants from adult mouse jejunum were cultured for 3 h in a medium which contained both ³H-fucose (10 or 25 Ci/ml) and monensin (100 M) or ³H-fucose only (control). Radiochemical analysis of cell fractions showed that ³H-fucose labelling of the brush border fraction decreased 42% in monensin-treated expiants, suggesting that in absorptive cells the intracellular transport of newly synthesized glycoproteins to the apical plasma membrane had been inhibited. Electron-microscopic examination of treated expiants revealed a variation in response to the drug from region to region. In some areas, both absorptive and goblet cells exhibited little alteration. In others, the Golgi cisternae of both absorptive and goblet cells were entirely replaced by large vacuoles, and in the latter cell type, the cisternae of the rough endoplasmic reticulum were greatly distended. Electron-microscopic radioautographic analysis showed that in absorptive and goblet cells exhibiting little morphological change, intracellular transport of newly synthesized glycoproteins was similar to that in controls. In regions where absorptive cells exhibited extensive Golgi modifications, intracellular transport remained normal in some cases; more often-however, there was a marked inhibition (over 70%) of transport of labelled glycoproteins to the apical surface. Transport to the basolateral membrane was never affected. In goblet cells exhibiting modifications of the Golgi apparatus and rough endoplasmic reticulum, no incorporation of ³H-fucose label in the Golgi apparatus occurred, suggesting a block of intracellular transport proximal to the site at which ³H-fucose is added. In absorptive cells, this does not appear to be the case, since the level of ³H-fucose incorporation in all treated cells remained similar to that in controls.     

Differential effects of monensin on the plant secretory pathway

The sodium ionophore monensin was used as an inhibitor of Golgifunction to study the secretory event in suspension-culturedsycamore cells. The morphological changes induced by monensinwere recorded. Concurrent studies of the disturbance causedby the drug were carried out by biochemical, cytochemical andimmunocytochemical techniques. Monensin induced accumulationof smooth and coated vesicles in the vicinity of the Golgi apparatus,swelling of Golgi cisternae and also provoked the formationof extracytoplasmic pockets between the plasma membrane andthe cell wall. Cytochemical tests for polysaccharides and immunolocalizationof secreted pectins, recognized by the monoclonal antibody JIM7, were performed, and the results indicated that the biosynthesisand/or processing machinery of polysaccharides was affectedby the drug. In contrast, immunolocalization of glycoproteins,recognized by the monoclonal antibodies JIM 84 and JIM 13 demonstratedthat the accumulation of vesicles in the cytoplasm were Golgiderived, and that the secretion of glycoproteins was not drasticallyaffected by monensin. Fluorography of radiolabelled proteinsdemonstrated that in this system the secretion of proteins wasnot qualitatively changed by monensin. The implications of theseresults are discussed in the light of the current hypotheseson the effects of monensin on the secretory pathway. Key words: Golgi apparatus, immunocytochemistry, monensin, secretion     

Effect of chemical carcinogens on the ultrastructure of cultured differentiating myogenic cells

The electron microscope studies have been carried out on primary monolayer tissue culture obtained from body tissues of rat and C3H mouse embryos. The cells of tissue culture were mainly myoblasts and fibroblast-like cells. The cultures were treated with two different carcinogenic substances--benz(a)-pyrene (BP) and methylnitrosonitroguanidine (MNNG). The changes were uniform and showed some alterations in formation of cell complexes, the inhibition of development and maturing of muscle elements, and distrophy of cytoplasmic organelles. The revealed distinction in morphological reaction of myogenic cells to the effect of BP and MNNG were wave-like myofibrillar structures in MNNG-treated cultures.     

Effect of social isolation on the ultrastructure of the dog brain

Ultrastructural changes in the central nucleus of the amygdalar body, field CA I of the hippocampus, piriform cortex, field 17 and field 7 have been studied in dogs, bred under conditions of social isolation. The changes are more numerous in emotiogenic structures. Similar ultrastructural rearrangements are revealed in the amygdalar body, hippocampus and in both fields of the neocortex. They are: neurons with different degree of chromatolysis, reactive changes in some organelles, terminals with agglutinated vesicles, or with their reduced number, increasing amount of synapses with a short active zone. At the same time, in the piriform gyrus vacuoles, membrane-like and osmiophilic inclusions, terminals with granular vesicles of various size are found more often.     

Effect of temperature and monensin on the level of intracellular sodium and pH in perfused RIF-1 cultured cells

The direct measurement of temperature in subcutaneously (sc) implanted tumors shows that the actual tumor temperature is by 3-4 degrees C lower than the normal body temperature. Thus, the temperatures usually used for tumor hyperthermia are in fact heating the sc-tumors from 33 to 37 degrees C. The temperature increase during the perfusion of radiation-induced fibrosarcoma (RIF-1) cells from 33 to 37 degrees C caused a reversible increase in intracellular 23Na ([Na+]i) NMR signal intensity by 50-60%. This heating significantly decreased the intracellular pH (pH) in 5 min, but it returned back to the baseline level during the heating period. The 3ATP/P(i) remained generally unchanged throughout the experiment. Monensin, an antitumoral drug and Na+ ionophor, increased [Na+]i by 20% during cell superfusion without heating. When combined, monensin did not increase the heating effect on [Na+]i. However, when monensin was added to the superfusion media, the [Na+]i level did not return to baseline during post-heating recovery, but instead started to increase again. Monensin did not significantly change pH(i) and betaATP/P(i). Our data and the literature show that monensin can accelerate the processes leading to the collapse of the transmembrane Na+ gradient and thus can increase the thermo-sensitivity of tumor cells.     

Effects of chronic nicotine on heteromeric neuronal nicotinic receptors in rat primary cultured neurons

Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [12?I]epibatidine ([12?I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 μM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, β2 and α5 subunits, but α2, α3, α6 and β4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the β2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.     

A cell-free analysis of the endocytic pathway

The molecular control of the endocytic pathway is poorly understood. To obtain this information requires the use of cell-free systems which faithfully recreate the various endocytic events as they occur in the intact cell. Here I describe our approach to elucidating the mechanism which controls the fusion between different vesicles on the pathway.     

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.     

Effect of hypokinesia on the ultrastructure of emotion-controlling structures in the rat brain

Hypokinesia lasting 40 and 90 days causes a number of ultramicroscopic reorganizations in neurons, synapses and glial elements of the singular cortex and the central amygdaloid nucleus. The longer the time of hypokinesia, the more numerous and variable the reorganizations become. For the amygdaloid body presence of neurons with various degree of chromatolysis, reactive and destructive changes of organells, agglutination and reduction in number of the synaptic vesicles, increasing number of synapses, possessing certain features of low functional activity are most specific. For the singular cortes are peculiar convolution of nuclear and cellular membranes, synaptic contact, increasing intercellular spaces, as well as changes in the structure of some axo-spine synapses, the spine apparatus including.     

Effect of naftidrofuryl on metabolism and survival of cultured neurons

The direct influence of Naftidrofuryl, a drug widely used for the treatment of cerebrovascular diseases, on the metabolism and survival of neurons was investigated in 8-day-old chick embryo forebrain cultures. Interaction of Naftidrofuryl with neurons resulted in the increase of the intracellular cyclic AMP levels. Naftidrofuryl stimulated deoxyglucose uptake and lactate production in a time- and dose-dependent fashion. These effects were observed after a few minutes following the beginning of the treatment with Naftidrofuryl. In parallel to the action on the metabolic activities, Naftidrofuryl was able to increase the survival rate of a significant proportion of the neuronal population. These data support the notion that Naftidrofuryl may act as a neuroprotective agent.