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

Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.     

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.     

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.     

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.     

Evidence for a clathrin-independent endocytic pathway for APP internalization in the neuronal somatodendritic compartment

1. Download : Download high-res image (267KB)
2. Download : Download full-size image

     

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.     

Lipid domains in the endocytic pathway

Whereas endosomes connect with both exocytic and endocytic organelle via extensive lipid and protein traffic, each endosome has a distinct lipid and protein composition. Recent observations suggest that different lipid membrane domains exist even in the same endosome. These lipid domains, together with low pH milieu, may present a variety of micro-environments to cargo molecules. Evidence is accumulating which suggests that the alteration of these lipid microdomains may be involved in a number of pathological conditions.     

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.     

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.     

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     

Establishment of neuronal polarity: lessons from cultured hippocampal neurons

In recent years we have learned a great deal about the molecular mechanisms underlying axonal elongation and navigation and the manner in which extracellular signals modify a growth cone's course of action. Yet, the mechanisms responsible for the earlier events of axonal and dendritic generation are just beginning to be understood. The recent advances in this exciting field highlight the importance of studies of cell migration and axonal elongation for our current understanding of the establishment of neuronal polarity.     

Oiling the wheels of the endocytic pathway

An ever more complete picture of the organization and function of the endocytic pathway is emerging. New mechanisms, and in particular lipid-based mechanisms that couple membrane dynamics and sorting, are being unraveled. But the final picture is still coming into focus as new membrane domains, cell entry pathways and compartments come into view. Of special interest are the recent findings that pathogenic agents, in contrast to scientists, seem to have long discovered how to subvert membrane specialization to their own advantage.     

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.     

Ordering of compartments in the yeast endocytic pathway

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t-SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t-SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.     

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.