Tunneling nanotubes: A novel pharmacological target for neurodegenerative diseases?

Diversiform ways of intercellular communication are vital links in maintaining homeostasis and disseminating physiological states. Among intercellular bridges, tunneling nanotubes (TNTs) discovered in 2004 were recognized as potential pharmacology targets related to the pathogenesis of common or infrequent neurodegenerative disorders. The neurotoxic aggregates in neurodegenerative diseases including scrapie prion protein (PrPSc), mutant tau protein, amyloid-beta (Aß) protein, alpha-synuclein (α-syn) as well as mutant Huntington (mHTT) protein could promote TNT formation via certain physiological mechanisms, in turn, mediating the intercellular transmission of neurotoxicity. In this review, we described in detail the skeleton, the formation, the physicochemical properties, and the functions of TNTs, while paying particular attention to the key role of TNTs in the transport of pathological proteins during neurodegeneration.

Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of cell types.

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.

Synaptic activity-induced glycolysis facilitates membrane lipid provision and neurite outgrowth.

The formation of neurites is an important process affecting the cognitive abilities of an organism. Neurite growth requires the addition of new membranes, but the metabolic remodeling necessary to supply lipids for membrane expansion is poorly understood. Here, we show that synaptic activity, one of the most important inducers of neurite growth, transcriptionally regulates the expression of neuronal glucose transporter Glut3 and rate-limiting enzymes of glycolysis, resulting in enhanced glucose uptake and metabolism that is partly used for lipid synthesis. Mechanistically, CREB regulates the expression of Glut3 and Siah2, the latter and LDH activity promoting the normoxic stabilization of HIF-1α that regulates the expression of rate-limiting genes of glycolysis. The expression of dominant-negative HIF-1α or Glut3 knockdown blocks activity-dependent neurite growth in vitro while pharmacological inhibition of the glycolysis and specific ablation of HIF-1α in early postnatal mice impairs the neurite architecture. These results suggest that the manipulation of neuronal glucose metabolism could be used to treat some brain developmental disorders.

The ATG16L1 risk allele associated with Crohn's disease results in a Rac1-dependent defect in dendritic cell migration that is corrected by thiopurines.

Thiopurines are commonly used drugs in the therapy of Crohn's disease, but unfortunately only show a 30% response rate. The biological basis for the thiopurine response is unclear, thus hampering patient selection prior to treatment. A genetic risk factor associated specifically with Crohn's disease is a variant in ATG16L1 that reduces autophagy. We have previously shown that autophagy is involved in dendritic cell (DC)-T-cell interactions and cytoskeletal regulation. Here we further investigated the role of autophagy in DC cytoskeletal modulation and cellular trafficking. Autophagy-deficient DC displayed loss of filopodia, altered podosome distribution, and increased membrane ruffling, all consistent with increased cellular adhesion. Consequently, autophagy-deficient DC showed reduced migration. The cytoskeletal aberrations were mediated through hyperactivation of Rac1, a known thiopurine target. Indeed thiopurines restored the migratory defects in autophagy-deficient DC. Clinically, the ATG16L1 risk variant associated with increased response to thiopurine treatment in patients with Crohn's disease but not ulcerative colitis. These results suggest that the association between ATG16L1 and Crohn's disease is mediated at least in part through Rac1 hyperactivation and subsequent defective DC migration. As this phenotype can be corrected using thiopurines, ATG16L1 genotyping may be useful in the identification of patients that will benefit most from thiopurine treatment.

Beyond Krabbe's disease: The potential contribution of galactosylceramidase deficiency to neuronal vulnerability in late-onset synucleinopathies.

New insights into the pathophysiological mechanisms behind late-onset neurodegenerative diseases have come from unexpected sources in recent years. Specifically, the group of inherited metabolic disorders known as lysosomal storage diseases that most commonly affect infants has been found to have surprising similarities with adult neurodegenerative disorders. Most notable has been the identification of Gaucher's disease as a comorbidity for Parkinson's disease. Prompted by the recent identification of neuronal aggregates of α-synuclein in another lysosomal storage disease, Krabbe's disease, we propose the idea that a similar connection exists between adult synucleinopathies and Krabbe's. Similarities between the two diseases, including the pattern of α-synuclein aggregation in the brain of the twitcher mouse (the authentic murine model of Krabbe's disease), changes to lipid membrane dynamics, and possible dysfunction in synaptic function and macroautophagy, underscore a link between Krabbe's disease and late-onset synucleinopathies. Silent GALC mutations may even constitute a risk factor for the development of Parkinson's in certain patients. More research is required to identify definitively any link and the validity of this hypothesis, but such a connection would prove invaluable for developing novel therapeutic targets for Parkinson's based on our current understanding of Krabbe's disease and for establishing new biomarkers for the identification of at-risk patients. © 2016 Wiley Periodicals, Inc.

Varying effects of EGF, HGF and TGFß on formation of invadopodia and invasiveness of melanoma cell lines of different origin.

The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers. Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM. We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor ß (TGFß) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment. All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites. In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastases. Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents. Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio). The F:G ratio remained unchanged or was even increased in metastatic cell lines treated with TGFß. Our findings indicate that the effects of stimulation with EGF, HGF and TGFß on melanoma cell invasiveness could depend on melanoma cell progression stage.

Changes in red blood cell membrane structure in G6PD deficiency: an atomic force microscopy study.

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency affects over 400 million people worldwide. The hemolytic anemia in G6PD deficiency is usually triggered by oxidative stress, but the mechanism remains uncertain. We have used atomic force microscopy for studying changes in red blood cell membrane and providing new insights on the mechanism. METHODS: G6PD activity assay and molecular genetic tests were used for molecular diagnosis. AFM was used to investigate alterations in the ultrastructure of G6PD deficient RBC membranes, the influence of different primaquine concentrations, and the protective effects of vitamin C. RESULT: Nine variants were identified from 33 G6PD deficient individuals. AFM imaging and quantitative analysis showed that G6PD deficient erythrocytes became heterogeneous and roughness measurements of erythrocyte membranes are increased. G6PD enzyme activity and different mutations may relate with roughness parameters. Furthermore, primaquine induces an increased roughness and height of erythrocyte membrane. Meanwhile, primaquine induces damages to erythrocytes which could be prevented by vitamin C treatment in normal RBCs but not in G6PD deficient erythrocytes. CONCLUSIONS: Our research may give valuable information about the status of G6PD deficient patients and explore the mechanism of hemolytic anemia.

HIV-1 reprograms the migration of macrophages.

Macrophages are motile leukocytes, targeted by HIV-1, thought to play a critical role in host dissemination of the virus. However, whether infection impacts their migration capacity remains unknown. We show that 2-dimensional migration and the 3-dimensional (3D) amoeboid migration mode of HIV-1-infected human monocyte-derived macrophages were inhibited, whereas the 3D mesenchymal migration was enhanced. The viral protein Nef was necessary and sufficient for all HIV-1-mediated effects on migration. In Nef transgenic mice, tissue infiltration of macrophages was increased in a tumor model and in several tissues at steady state, suggesting a dominant role for mesenchymal migration in vivo. The mesenchymal motility involves matrix proteolysis and podosomes, cell structures constitutive of monocyte-derived cells. Focusing on the mechanisms used by HIV-1 Nef to control the mesenchymal migration, we show that the stability, size, and proteolytic function of podosomes are increased via the phagocyte-specific kinase Hck and Wiskott-Aldrich syndrome protein (WASP), 2 major regulators of podosomes. In conclusion, HIV-1 reprograms macrophage migration, which likely explains macrophage accumulation in several patient tissues, which is a key step for virus spreading and pathogenesis. Moreover, Nef points out podosomes and the Hck/WASP signaling pathway as good candidates to control tissue infiltration of macrophages, a detrimental phenomenon in several diseases.

Recruitment of Nox4 to a plasma membrane scaffold is required for localized reactive oxygen species generation and sustained Src activation in response to insulin-like growth factor-I.

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.

Changes in red blood cell membrane structure in type 2 diabetes: a scanning electron and atomic force microscopy study.

Red blood cells (RBCs) are highly deformable and possess a robust membrane that can withstand shear force. Previous research showed that in diabetic patients, there is a changed RBC ultrastructure, where these cells are elongated and twist around spontaneously formed fibrin fibers. These changes may impact erythrocyte function. Ultrastructural analysis of RBCs in inflammatory and degenerative diseases can no longer be ignored and should form a fundamental research tool in clinical studies. Consequently, we investigated the membrane roughness and ultrastructural changes in type 2 diabetes. Atomic force microscopy (AFM) was used to study membrane roughness and we correlate this with scanning electron microscopy (SEM) to compare results of both the techniques with the RBCs of healthy individuals. We show that the combined AFM and SEM analyses of RBCs give valuable information about the disease status of patients with diabetes. Effectiveness of treatment regimes on the integrity, cell shape and roughness of RBCs may be tracked, as this cell's health status is crucial to the overall wellness of the diabetic patient.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction.

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind-ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell-cell or bridging MNTs and blind-ended MNTs was greatest between major histocompatibility complex (MHC) class II(+) cells during corneal injury or TLR ligand-mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid-derived cells in inflamed corneal explants from Cx(3)cr1(GFP) and CD11c(eYFP) transgenic mice revealed that MNTs form de novo at a rate of 15.5 µm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell-cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus-1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

In vivo laser confocal microscopic analysis of corneal K-structures after keratorefractive surgery (LASIK and epi-LASIK).

BACKGROUND AND OBJECTIVE: To demonstrate alterations of corneal K-structures (sub-Bowman's fibrous structures) after keratorefractive surgery by in vivo laser confocal microscopy and to look for association of K-structures with fluorescein-stained anterior corneal mosaic (ACM). PATIENTS AND METHODS: Five patients (nine eyes) participated in this study. For four patients, one eye was evaluated after laser in situ keratomileusis (LASIK) and the other after epipolis-laser in situ keratomileusis (epi-LASIK). For one patient, the left eye was evaluated after epithelial debridement. A photograph of the ACM was obtained. Central corneal regions were scanned by Heidelberg Retina Tomograph 2 Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany). RESULTS: The ACM and K-structures disappeared in all corneas after epi-LASIK, but not after LASIK and epithelial debridement cornea. CONCLUSION: The presence of K-structures and ACM may be an index to identify eyes that had a previous refractive surgical procedure (surface ablation or LASIK) and be a health index of Bowman layer and adjacent anterior stroma.

Globotriaosyl ceramide receptor function - where membrane structure and pathology intersect.

The glycosphingolipid globotriaosyl ceramide, (Galalpha1-4Galss1-4 glucosyl ceramide-Gb(3)) also known as CD77 and the P(k) blood group antigen, is bound by both verotoxins and by the HIV adhesin, gp120. Gb(3) plays an important receptor role in VT induced hemolytic uremic syndrome (HUS) and HIV infection. The organization of glycolipids, including Gb(3), into lipid rafts is central to both pathologies. The fatty acid heterogeneity within the Gb(3) lipid moiety plays a central role in assembly within such ordered domains. Differential binding of verotoxins and gp120 to such Gb(3) isoforms in model and cell membranes indicates a significant role in the eventual pathogenic outcome. HUS may provide the first example whereby membrane Gb(3) organization provides a predictor for tissue selective in vivo pathology.

Circulating tissue factor-exposing microparticles.

Upon stimulation or apoptosis, eukaryotic cells shed membrane vesicles of submicron size. These so-called microparticles (MPs) are detected and characterized based on the exposure of antigens characteristic of their respective parental cells and on the increased distribution of negatively charged phospholipids to the outer membrane layer. Among the various hypothesized functions of MPs in both health and disease, one of the most studied is their possible role in hemostasis and thrombosis. In this context, special attention is paid to tissue factor (TF) exposed on a variety of MPs. MPs may have outstanding functional because of their ability to display "active" TF due to an abundance of negatively charged phospholipids on their surface. The rapid accumulation of TF-bearing MPs (TF+MPs) in a developing thrombus as well as the increased numbers and thrombogenic activity of TF+MPs in prothrombotic disorders indicate an important role in the pathogenesis of thrombosis. Nevertheless, isolation, quantification and antigenic characterization of TF+MPs proved challenging and a lively scientific debate is ongoing with respect to a reliable method to determine the cellular source of MP in vivo. Standardization of preanalytical procedures and development of more sensitive technologies are needed to improve our current understanding of the role of circulating TF+MPs in thrombosis.

Microparticles in vascular diseases.

Cellular microparticles (MP) are small membrane vesicles that are released from cells upon activation or apoptosis. They constitute a heterogeneous population of submicron elements differing in cellular origin, number, size, antigenic composition and functional properties. Circulating MP provide an additional procoagulant phospholipid surface enabling the assembly of the clotting enzyme complexes and thrombin generation. Their procoagulant properties rely on the exposure of phosphatidylserine and on the possible presence of tissue factor, the main initiator of blood coagulation. Microparticles constitute the main reservoir of blood-borne tissue factor. Derived from various cells, most notably platelets, erythrocytes, leucocytes and endothelial cells, circulating MP are detectable in the circulation of healthy subjects. Elevated levels are encountered in diseases with vascular involvement and hypercoagulability such as disseminated intravascular coagulation, diabetes, immune-mediated thrombosis, kidney diseases, acute coronary syndromes or systemic inflammatory disease, where they appear indicative of a poor clinical outcome. Converging evidence from experimental and clinical data underlines an involvement of procoagulant MP in the initiation/dissemination of procoagulant and inflammatory responses. In these clinical settings, the pharmacological modulation of MP level or activity provides challenging issues.

Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes.

Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ET(A))/ET type B (ET(B)) receptor family. ET(A), the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ET(A) in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ET(A) protein levels. In these T tubule-compromised myocytes, an overexpression of ET(A) using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ET(A) downregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ET(A) and intact T tubules (preserved with CD). Therefore, the activation of ET(A) localized in T tubules was associated with a strong PIE, whereas the activation of ET(A) in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ET(A) is upregulated and T-tubule morphology is altered.

Platelet microparticles and vascular cells interactions: a checkpoint between the haemostatic and thrombotic responses.

Described 40 years ago as cell dust, microparticles (MPs) are now considered a key component in the haemostatic response. Owing to their plasma membrane reactivity, platelets are believed to constitute the main source of circulating procoagulant microparticles and behave as true sensors for the haemostatic response. Erythrocytes, leukocytes and endothelial cells are also able to shed MPs in the blood flow, their respective contribution varying with the pathophysiologic circumstances and extent of the cellular damage. The catalytic properties of MPs rely on a procoagulant anionic phospholipid, phosphatidylserine, made accessible at the outer leaflet following plasma membrane remodelling and on the eventual presence of tissue factor (TF). Under resting conditions, most membrane-bound TF is encrypted. Although able to bind to FVIIa, it does not trigger blood coagulation. Under prothrombotic conditions, TF decryption would occur through intricate pathways involving platelets, monocytes, endothelial cells and derived MPs. P-selectin/P-selectin glycoprotein Ligand-1 (PSGL-1) interactions and reactive oxygen species would promote TF decryption in cell-MP aggregates. At sites of endothelium injury, the swift recruitment of TF+-MPs through P-selectin/PSGL-1 interactions enables the concentration of TF activity above a threshold allowing coagulation to be triggered. Another crucial feature in the initiation of blood coagulation, possibly tuned by MPs, is the balance between TF and TFPI. In specific pathophysiologic contents with elevated levels of circulating TF+-MPs, accessible TFPI at the MP surface would be overwhelmed. Beyond their procoagulant properties demonstrated in vitro, a number of pieces of evidence points to procoagulant MPs as efficient effectors in the haemostatic response, and as pathogenic markers of thrombotic disorders and vascular damage. This review will focus on the pathophysiological significance of platelet-derived MPs and their interaction with vascular cells.

Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure.

Endothelial dysfunction and arterial stiffness are major determinants of cardiovascular risk in patients with end-stage renal failure (ESRF). Microparticles are membrane fragments shed from damaged or activated cells. Because microparticles can affect endothelial cells, this study investigated the relationship between circulating microparticles and arterial dysfunction in patients with ESRF and identified the cellular origin of microparticles associated with these alterations. Flow cytometry analysis of platelet-free plasma from 44 patients with ESRF indicated that circulating levels of Annexin V+ microparticles were increased compared with 32 healthy subjects, as were levels of microparticles derived from endothelial cells (three-fold), platelets (16.5-fold), and erythrocytes (1.6-fold). However, when arterial function was evaluated noninvasively in patients with ESRF, only endothelial microparticle levels correlated highly with loss of flow-mediated dilation (r = -0.543; P = 0.004), increased aortic pulse wave velocity (r = 0.642, P < 0.0001), and increased common carotid artery augmentation index (r = 0.463, P = 0.0017), whereas platelet-derived, erythrocyte-derived, and Annexin V+ microparticle levels did not. In vitro, microparticles from patients with ESRF impaired endothelium-dependent relaxations and cyclic guanosine monophosphate generation, whereas microparticles from healthy subjects did not. Moreover, in vitro endothelial dysfunction correlated with endothelial-derived (r = 0.891; P = 0.003) but not platelet-derived microparticle concentrations. In fact, endothelial microparticles alone decreased endothelial nitric oxide release by 59 +/- 7% (P = 0.025). This study suggests that circulating microparticles of endothelial origin are tightly associated with endothelial dysfunction and arterial dysfunction in ESRF.