Mitochondrial involvement in sensory neuronal cell death and survival

Peripheral nerve injuries (PNI) are continuing to be an ever-growing socio-economic burden affecting mainly the young working population and the current clinical treatments to PNI provide a poor clinical outcome involving significant loss of sensation. Thus, our understanding of the underlying factors responsible for the extensive loss of the sensory cutaneous subpopulation in the dorsal root ganglia (DRG) that occurs following injury needs to be improved. The current investigations focus in identifying visual cues of mitochondria-related apoptotic events in the various subpopulations of sensory cutaneous neurons. Sensory neuronal subpopulations were identified using FastBlue retrograde labelling following axotomy. Specialised fluorogenic probes, MitoTracker Red and MitoTracker Orange, were employed to visualise the dynamic changes of the mitochondrial population of neurons. The results reveal a fragmented mitochondrial network in sural neurons following apoptosis, whereas a fused elongated mitochondrial population is present in sensory proprioceptive muscle neurons following tibial axotomy. We also demonstrate the neuroprotective properties of NAC and ALCAR therapy in vitro. The dynamic mitochondrial network breaks down following oxidative exposure to hydrogen peroxide (H₂O₂), but reinitiates fusion after NAC and ALCAR therapy. In conclusion, this study provides both qualitative and quantitative evidence of the susceptibility of sensory cutaneous sub-population in apoptosis and of the neuroprotective effects of NAC and ALCAR treatment on H₂O₂-challenged neurons.     

Betanodavirus: Mitochondrial disruption and necrotic cell death

Betanodaviruses cause viral nervous necrosis, an infectious neuropathological condition in fish that is characterized by necrosis of the central nervous system, including the brain and retina. This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance. The mechanism of brain and retina damage during betanodavirus infection is poorly understood. In this review, we will focus recent results that highlight betanodavirus infection-induced molecular death mechanisms in vitro. Betanodavirus can induce host cellular death and post-apoptotic necrosis in fish cells. Betanodavirus-induced necrotic cell death is also correlated with loss of mitochondrial membrane potential in fish cells, as this necrotic cell death is blocked by the mitochondrial membrane permeability transition pore inhibitor bongkrekic acid and the expression of the anti-apoptotic Bcl-2 family member zfBcl-xL. Moreover, this mitochondria-mediated necrotic cell death may require a caspase-independent pathway. A possible cellular death pathway involving mitochondrial function and the modulator zfBcl-xs is discussed which may provide new insights into the necrotic pathogenesis of betanodavirus.     

Mitochondrial fission proteins regulate programmed cell death in yeast

The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.     

Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes

Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.     

Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death

Cigarette smoke-induced apoptosis and necrosis contribute to the pathogenesis of chronic obstructive pulmonary disease. The induction of heme oxygenase-1 provides cytoprotection against oxidative stress, and may protect in smoking-related disease. Since mitochondria regulate cellular death, we examined the functional expression and mitochondrial localization of heme oxygenase-1 in pulmonary epithelial cells exposed to cigarette smoke extract (CSE), and its role in modulating cell death. Heme oxygenase-1 expression increased dramatically in cytosolic and mitochondrial fractions of human alveolar (A549), or bronchial epithelial cells (Beas-2b) exposed to either hemin, lipopolysaccharide, or CSE. Mitochondrial localization of heme oxygenase-1 was also observed in a primary culture of human small airway epithelial cells. Furthermore, heme oxygenase activity increased dramatically in mitochondrial fractions, and in whole cell extracts of Beas-2b after exposure to hemin and CSE. The mitochondrial localization of heme oxygenase-1 in Beas-2b was confirmed using immunogold-electron microscopy and immunofluorescence labeling on confocal laser microscopy. CSE caused loss of cellular ATP and rapid depolarization of mitochondrial membrane potential. Apoptosis occurred in Beas-2b at low concentrations of cigarette smoke extract, whereas necrosis occurred at high concentrations. Overexpression of heme oxygenase-1 inhibited CSE-induced Beas-2b cell death and preserved cellular ATP levels. Finally, heme oxygenase-1 mRNA expression was elevated in the lungs of mice chronically exposed to cigarette smoke. We demonstrate the functional compartmentalization of heme oxygenase-1 in the mitochondria of lung epithelial cells, and its potential role in defense against mitochondria-mediated cell death during CSE exposure.     

Mitochondria and their role in cell metabolism

Mitochondria are subcellular organelles of the endosymbiotic origin. They are bounded by double membrane and contain their own DNA. Recent advance in 3D microscopy have contributed a better understanding of mitochondrial structure. Mitochondria are highly dynamic organelles with a very complex structure of the inner membrane. In cells, mitochondria create an interconnected reticulum. Beyond a fundamental role in energy production, they also play key roles in thermogenesis, maintenance of cellular redox potential, Ca2+ homeostasis, ROS production, cell signaling and cell death. Disturbances in mitochondrial metabolism are known to play a role not only in rare genetics disorders, but have also been implicated in many common diseases of aging. Conventional studies of mitochondrial metabolism are based on the isolation of intact organelles. Because of mitochondrial complex roles rises a need to assay mitochondrial functions in situ. The activity of respiration and oxidative phosphorylation in intact and permeabilized cells can be measured by using high resolution respirometry. We can estimate various mitochondrial functions in living cells by using fluorescent cation dyes.     

The role of TRPM channels in cell death

Transient receptor potential (TRP) channels of the melastatin-like family (TRPM) play critical roles in mediating cellular responses to a wide range of physiological stimuli that, under certain situations, can induce cell death. To date, two TRPM family members, TRPM2 and TRPM7, have been implicated directly as central components of cell death pathways. TRPM2, a Ca²⁺-permeant, non-selective cation channel, senses and responds to oxidative stress levels in the cell. TRPM7 is required for cell viability and has been proposed recently to mediate stress-induced cell death in the central nervous system. We review here the evidence for the involvement of these TRPM channels in cell death processes and discuss the mechanisms by which TRPM channel activation occurs. The ability to attenuate expression levels and functionality of these channels is necessary to understand the involvement of TRPM in cell death and we evaluate current approaches for modulation of TRPM channel function. Finally, we discuss the possibility that TRPM channels may provide therapeutic targets for degenerative diseases involving oxidative stress-related pathologies including diabetes and Alzheimers disease.     

细胞凋亡的线粒体途径与缺血缺氧性肾损伤

“外源性通路”和“内源性通路”是细胞内凋亡信号转导的两条基本途径。线粒体作为内源性通路的中心环节在其中起主导作用。细胞凋亡时线粒体膜通透性增加,释放可溶性膜间隙蛋白（细胞色素,凋亡诱导因子,Smac／DIABLO等）,启动caspase级联反应和不依赖caspase途径的方式参与凋亡发生。Bcl-2蛋白家族以线粒体为靶位调控凋亡。线粒体凋亡途径也参与了肾缺血再灌注的损伤,其中炎症反应与细胞凋亡发生交互作用使得其损伤机制更为复杂。     

Mitochondrial membrane potential and ischemic neuronal death

Mitochondria are intracellular organelles in which high energy phosphate is produced. Ischemia causes depletion of the materials necessary to produce this phosphate and strongly affects the electron transport chain. Apoptosis commences during and after ischemia. As such, it is likely that a significant relationship exists between inactivation of electron transport and apoptosis. Mitochondrial membrane potential (MMP) reflects performance of the electron transport chain and can indicate a pathological disorder of this system. In an experimental setting, oxygen-glucose depletion (OGD) in neuronal cell culture has been employed to simulate an ischemic condition. The relationship between MMP and subsequent neuronal death during and after OGD has been examined. MMP dissipation and concomitant neuronal death have been reported, but recent studies have demonstrated mitochondrial hyperpolarization preceding neuronal death. The direction of MMP polarization depends on the extent of OGD. Long OGD results in depolarization, while shorter OGD induces hyperpolarization. Neurons are still viable during hyperpolarization, but the process may switch on the apoptotic cascade. Meanwhile, dissipation of MMP seems to be a consequence of severe energy deficit, leading to necrosis. MMP may be a marker of subsequent apoptosis, although a causal relationship remains to be determined.     

Induced cell death and its role in hemopoietic hypoplasia after cytostatic treatment

The morphological signs of apoptosis in the bone-marrow cells and thymocytes, indices of cellularity in these organs and in peripheral blood and the absolute number of committed bone marrow cells-precursors have been studied on CBA mice injected with Etoposid (1/2 LD₅₀). The results of the study suggest that reduced cellular counts observed in the hemopoietic organs 3–6 h after the cytostatic injection are due to Etoposid-induced apoptosis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 39–42, January 1999     

The emerging role of STING-dependent signaling on cell death

Immunologic Research - STING is a newly identified adaptor protein for sensing cytosolic nucleic acid. It is well established that STING plays a crucial role in innate immune response via inducing...     

Cyclic modulation of sertoli cell junctional complexes in a seasonal breeder: The mink (Mustela vison)

The development and modulation of Sertoli cell junctions was studied in newborn and adult mink during the active and inactive spermatogenic phases. The techniques used were electron microscopy of freeze-fractured replicas and thin sections of tissues infused with horseradish peroxidase as a junction permeability tracer. In the newborn, freeze-fractured developing junctions had either spherical or fibrillar particles. In addition, Junctional domains where particles were associated preferentially with the E-face, and others where particles were associated preferentially with the P-face, were found developing either singly or conjointly within a given membrane segment, thus yielding a heterogeneous Junctional segment. Coincidently with the development of a tubular lumen and the establishment of a competent blood-testis barrier, Junctional strands were composed primarily of particulate elements associated preferentially with the E-face. In adult mink during active spermatogenesis, cell junctions were found on the entire lateral Sertoli cell plasma membrane from the basal to the luminal pole of the cell. In the basal third of the Sertoli cell, membranous segments that faced a spermatogonium or a migrating spermatocyte displayed forming tight, gap, and adherens junctions. In the middle third, abutting membrane segments localized above germ cells were involved in continuous zonules and in adherens junctions. In the apical or luminal third, the zonules were discontinuous, and the association of Junctional particles with the E-face furrow was lost. Gap junctions increased in both size and numbers. Junctional vesicles that appeared as annular gap and tight-junction profiles in thin sections or as hemispheres in freeze-fracture replicas were present. Reflexive tight and gap junctions were formed through the interaction of plasma membrane segments of the same Sertoli cell. Internalized Junctional vesicles were also present in mature spermatids. During the inactive spermatogenic phase, cell junctions were localized principally in the basal third of the Sertoli cell; Junctional strands resembled those of the newborn mink. During the active spermatogenic phase, continuous zonules were competent in blocking passage of the protein tracer. During the inactive phase the blood-testis barrier was incompetent in blocking entry of the tracer into the seminiferous epithelium. It is proposed that modulation of the Sertoli cell zonules being formed at the base and dismantled at the apex of the seminiferous epithelium follows the direction of germ cell migration and opposes the apicobasal direction of junction formation reported for most epithelia. The arrest of spermatogenesis coincides with dramatic changes in the dynamic modifications of Sertoli cell zonules.     

Central role of lactic acidosis in cancer cell resistance to glucose deprivation-induced cell death

Solid tumours are dependent on glucose, but are generally glucose-deprived due to poor vascularization. Nevertheless, cancer cells can generally survive glucose deprivation better than their normal counterparts. Thus, to render cancer cells sensitive to glucose depletion may potentially provide an effective strategy for cancer intervention. We propose that lactic acidosis, a tumour microenvironment factor, may allow cancer cells to develop resistance to glucose deprivation-induced death, and that disruption of lactic acidosis may resume cancer cells' sensitivity to glucose depletion. Lactic acidosis, lactosis, or acidosis was generated by adding pure lactic acid, sodium lactate, or HCl to the culture medium. Cell death, cell cycle, autophagy, apoptosis, and gene expression profiling of the surviving cancer cells under glucose deprivation with lactic acidosis were determined. Under glucose deprivation without lactic acidosis, 90% of 4T1 cancer cells died within a single day; in a sharp contrast, under lactic acidosis, 90% of 4T1 cells died in a period of 10 days, with viable cells identified even 65 days after glucose was depleted. Upon glucose restoration, surviving cells resumed proliferation. Lactic acidosis also significantly extended survival of other cancer cells under glucose deprivation. G1/G0 arrest, autophagy induction, and apoptosis inhibition were tightly associated with lactic acidosis-mediated resistance to glucose deprivation. Lactosis alone had no effect on cell survival under glucose deprivation; acidosis alone can prolong cell survival time but is not as potent as lactic acidosis. Thus, the ability of cancer cells to resist glucose deprivation-induced cell death is conferred, at least in part, by lactic acidosis, and we envision that disrupting the lactic acidosis may resume the sensitivity of cancer cells to glucose deprivation.     

Spermiogenesis in Nautilus pompilius. II. Sertoli cell-spermatid junctional complexes

A "ball and socket-like" junction between branches of the Sertoli cells and the developing spermatids is described. A cytoplasmic extension of the Sertoli cell fits into a pocket in the spermatid and has a constricted neck region. Frequently there are large multivesiculate bodies in the Sertoli cell extension and small vesicles frequently appear in the spermatid cytoplasm, in the area of the "ball and socket-like" junction. This suggests the possibility that there may be communication of materials between the two cells. The possible function of the junctions is discussed and it is concluded that they more likely have a role in nutrition than in coordination.     

Intercellular junctional specializations in human basal cell carcinoma

Summary Intercellular junctions of various types were found on the membrane fracture faces of human nodular basal cell carcinoma (BCC) cells. The junctional types represented include desmosomes, tight junctions, and gap junctions. A semiquantitative comparison of undifferentiated and differentiated nodular BCC showed that gap and tight junctions were observed on all exposed membrane fracture interfaces of the differentiated tumors, while only fifty six per cent of the membrane interfaces of the undifferentiated tumor exhibited similar junctional specializations. These membrane specializations may be a partial reflection of differentiation among the different types of BCC and their contribution to the less invasive character of nodular BCC cannot be ruled out.Preliminary results from this study were presented at the X Triennial World Congress of Pathology Sept. 25–29, 1978, Rio de Janeiro, Brazil     

The functional role of stress proteins in ER stress mediated cell death

The endoplasmic reticulum (ER) is an intracellular organelle involved in biosynthesis and the secretory pathway. This organelle has many resident proteins including biosynthetic enzymes and secretory proteins. Recent studies have suggested that dysfunction of the ER or secretory pathway is involved in the pathogenesis of various human diseases. Some stresses acting on the ER, which are designated ER stress, induce the accumulation of unfolded/misfolded proteins in the ER, leading to cell death. Misfolded proteins are retained until they form their native conformation or returned to the cytosol for degradation by the proteasome. Among the ER-resident proteins, molecular chaperones prevent aggregation of proteins within the ER, and orchestrate the ER quality control systems. We have reported the roles of novel stress proteins, namely 150-kDa oxygen-regulated protein, 94-kDa glucose-regulated protein and RA410. These proteins are induced significantly by hypoxia or oxidative stress and have cytoprotective effects under these conditions. These findings suggest that hypoxia and oxidative stress target the ER and secretory pathway, resulting in ER stress, and that these proteins exert cytoprotective effects in various diseases associated with ER stress.     

The role of activation-induced cell death in the differentiation of T-helper-cell subsets

Activation-induced cell death (AICD) has been demonstrated in T-cell hybridomas, immature thymocytes, and activated mature T cells. However, the molecular mechanisms of AICD and its physiological role in T-helper-cell differentiation remain uncertain. Recently, we have shown that Th1 and Th2 cells have distinct mechanisms of AICD. Our findings suggest that signaling from cytokines initiates the differentiation program, but that the selective action of death effectors determines the fate of differentiating T-helper cells, and thus, the ultimate balance between T-helper subpopulations. Among T cells, activation-induced expression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is observed exclusively in Th2 clones and primary T-helper cells differentiated under Th2 conditions, while the expression of CD95L (Fas ligand) occurs mainly in Th1 cells. Furthermore, Th1 cells are more susceptible than Th2 cells to apoptosis induced through either TRAIL or CD95L, and radiolabeled Th1 cells can be induced into apoptosis via fratricide by both Th1 and Th2 cells, while Th2 cells are spared. The pan-caspase inhibitor, z-VAD, prevents AICD in Th1 cells, but not Th2 cells, indicating different mechanisms of AICD in each T-helper subtype. Antibody blockade of TRAIL and CD95L significantly boosts interferon-γ (IFN-γ) production in vitro. Also, young mice with mutant CD95 (MRL/MpJ-lpr/lpr) have a stronger Th1 response to ovalbumin immunization than do controls. We conclude that apoptosis mediated by CD95L and TRAIL is critical in the selective removal of differentiating T helper cells.     

Dexamethasone preferentially suppresses plasmacytoid dendritic cell differentiation and enhances their apoptotic death

Plasmacytoid dendritic cells (pDC) are an important source of type-1 interferon (IFN) following microbial infection and also play key roles in the induction of innate and adaptive immune responses. Here, we show that the glucocorticoid (GC) dexamethasone (Dex) strikingly reduces pDC (and myeloid DC) numbers in secondary lymphoid tissue and liver of normal and hematopoietic growth factor-mobilized mice and suppresses pDC differentiation from bone marrow precursors in vitro. Moreover, the apoptotic death of pDC in vitro was enhanced by exposure to Dex. Notably, however, Toll-like receptor 9 expression and virally induced IFNalpha production by residual pDC from Dex-treated animals were unaffected. Thus, whereas marked reduction in absolute numbers of pDC by GC may predispose to viral infection, often associated with GC-mediated immunosuppression, reductions in pDC and IFNalpha production may contribute to the beneficial effects on GC observed in systemic autoimmune disease, in which that both pDC and IFNalpha have been implicated.