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VAMP8 deficiency led to increased rates of apoptotic thymocytes. a–c TUNEL staining of apoptotic cells in thymus sections revealed a high number of apoptotic cells in a small and sick Vamp8−/− mouse (12 days old, a) compared with a heterozygous mouse (8 days old, b). c A small and healthy Vamp8−/− littermate did not display increased apoptosis. Bars 20 µm. d Thymocytes of Vamp8−/− mice and heterozygous littermates were stained with annexin V and propidium iodide. Summary of the proportion of annexin-V- and propidium-iodide-negative viable cells among thymocytes from small and sick (n = 4), small and healthy (n = 3), and adult (n = 6) Vamp8−/− mice and their heterozygous littermates is shown (bars percentage of viable thymocytes ±SD). The small and sick Vamp8−/− mice had significantly fewer viable thymocytes than their heterozygous littermates (n = 4, *P = 0.02, U test). e In a flow cytometric analysis, the percentage of annexin-V-positive apoptotic thymocytes was determined among the four major subpopulations defined by CD4 and CD8 markers (bars percentage of apoptotic thymocytes 1+SD as determined in small and healthy Vamp8−/− mice and their heterozygous littermates; n = 3)
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SNARE (soluble-N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the recognition and fusion of transport vesicles in eukaryotic cells. The SNARE protein VAMP8 (also called endobrevin) is involved in the fusion of late endosomes and in some pathways of regulated exocytosis. In a subset of mice deficient for the SNARE protein VA...
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... We investigated the role of a TRIM6-dependent gene not previously described to regulate WNV replication or IFN-I signaling, Vamp8. VAMP8 is a vesicleassociated-membrane protein in the SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein) family known to modulate endocytosis (32), exocytosis of secretory (33)(34)(35)(36) and lytic (37) granules, thymus development (38), receptor exocytosis (39), and cross-presentation by antigen-presenting cells (40). We found that VAMP8 does not directly affect WNV replication but promotes optimal IFN-I signaling. ...
WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.
... This reduction in lipid peroxidation was similar to what we observed previously with siRNA knockdown of gp91 phox , or upon treatment of the DCs with the NOX2-inhibitor phenylarsine oxide or the lipophilic radical scavenger α-tocopherol . Lipid peroxidation was also reduced in BMDCs isolated from VAMP8-/-mice compared to BMDCs from wild-type mice ( Fig. 2F; Kanwar et al., 2008;Matheoud et al., 2013), although in this case LPS maturation only resulted in increased surface expression of CD86 and did not affect MHC class I, MHC class II and CD11c levels (Sup. Fig. 1B). ...
Cross-presentation of foreign antigen in major histocompatibility complex (MHC) class I by dendritic cells (DCs) requires activation of the NADPH-oxidase NOX2 complex. We recently showed that NOX2 is recruited to phagosomes by the SNARE protein VAMP8 where NOX2-produced reactive oxygen species (ROS) cause lipid oxidation and membrane disruption, promoting antigen translocation into the cytosol for cross-presentation. In this study, we extend these findings by showing that VAMP8 is also involved in NOX2 trafficking to endosomes. Moreover, we demonstrate in both human and mouse DCs that absence of VAMP8 leads to decreased ROS production, lipid peroxidation and antigen translocation, and that this impairs cross-presentation. In contrast, knockdown of VAMP8 did not affect recruitment of MHC class I and the transporter associated with antigen processing 1 (TAP1) to phagosomes, although surface levels of MHC class I were reduced. Thus, in addition to a secretory role, VAMP8-mediates trafficking of NOX2 to endosomes and phagosomes and this promotes induction of cytolytic T cell immune responses.
... The larger subset in community B has only one high-hub, SNAP23, which stabilizes SNARE complexes orchestrating ERC-phagosome fusion and enrichment of phagosomes with ERC-derived MHC-I [145]. Moreover, SNARE protein VAMP8 interacts with SNAP23 and has a specific function in the thymic stroma affecting the proliferation and apoptosis of T-lymphocytes during maturation in the thymus [146]. There are three VIPs in the larger subset of community B. One is ADNP2, which codes for the Activity-dependent Neuroprotective Protein (ADNP) and is probably involved in protecting developing thymocytes from apoptosis, being regulated by the vasoactive intestinal peptide [147,148,149,150]. ...
Trisomy 21-driven transcriptional alterations in human thymus were characterized through gene coexpression network (GCN) and miRNA-target analyses. We used whole thymic tissue - obtained at heart surgery from Down syndrome (DS) and karyotipically normal subjects (CT) - and a network-based approach for GCN analysis that allows the identification of modular transcriptional repertoires (communities) and the interactions between all the system's constituents through community detection. Changes in the degree of connections observed for hierarchically important hubs/genes in CT and DS networks corresponded to community changes. Distinct communities of highly interconnected genes were topologically identified in these networks. The role of miRNAs in modulating the expression of highly connected genes in CT and DS was revealed through miRNA-target analysis. Trisomy 21 gene dysregulation in thymus may be depicted as the breakdown and altered reorganization of transcriptional modules. Leading networks acting in normal or disease states were identified. CT networks would depict the "canonical" way of thymus functioning. Conversely, DS networks represent a "non-canonical" way, i.e., thymic tissue adaptation under trisomy 21 genomic dysregulation. This adaptation is probably driven by epigenetic mechanisms acting at chromatin level and through the miRNA control of transcriptional programs involving the networks' high-hierarchy genes.
... Alternatively, VAMP8 deficiency may lead to a disparate clinical presentation. Notably, one third of Vamp8 knockout mice die before the age of 1 mo (Wang et al., 2004;Kanwar et al., 2008). By comparison, Prf1 knockout mice are healthy until challenged by viral infection (Kägi et al., 1994), suggesting that VAMP8 mediates vital physiological functions in addition to lymphocyte cytotoxicity. ...
Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.
... CD95, promoted CD95 receptor clustering and internalization, caspase activation and loss of mitochondrial membrane potential that are the main processes involved in apoptosis [115]. VAMP-8 has a unique function in the thymic stroma affecting the proliferation and apoptosis of T lymphocytes during maturation in the thymus [116]. The novel role of α-SNAP in tumour progression and anti-apoptotic function has been investigated. ...
... Several knockout mice showing dwarfism and a small thymus have been reported [3,6,8]. In latent transforming growth factor-b binding protein (Ltbp)-3 null mice displaying growth retardation with multiple skeletal anomalies, defective thymus seems to be attributable to an involution rather than congenital hypoplasia because the mice show elevated corticosterone levels in the serum [3]. ...
... Thymic atrophy, accompanied with lymphopenia, is caused by the enhancement of BMP-4 signaling [8]. In mice deficient for VAMP8/endobrevin, which is associated with soluble-N-ethylmaleimide-sensitive factor attachment receptor (SNARE), the organization of the thymic epithelium and T cell development were severely affected in the mice [6]. Common pathological alterations in the thymuses of these knockout mice and pet/pet rats are reduced proliferation and increased apoptosis in their thymocytes. ...
The petit rat (pet/pet) is a new semi-lethal dwarf mutant with anomalies in the thymus and testes, defects inherited as a single autosomal recessive trait. At birth, these pet/pet rats show low birth weight and extremely small thymuses; at 140 days of age, their thymuses show abnormal involution. In the present study, we examined early postnatal development of hypoplastic pet/pet thymuses. In addition to being hypoplastic at birth, pet/pet thymus growth was almost completely impaired during the early postnatal period. As shown by cellular incorporation of BrdU, the mitotic activity was lower in pet/pet than in normal thymuses, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that apoptosis occurred more often in pet/pet than in normal thymus cells during the first few days after birth. These results indicate that postnatal development of the hypoplastic pet/pet thymus is defective due to the reduced proliferation and increased apoptosis of thymic cells.
... In contrast, the numbers of TUNEL-positive cells in the medulla were comparable in pet/pet and normal thymuses at all time points (data not shown). Several knockout mice showing dwarfism and a small thymus have been reported [3, 6, 8]. In latent transforming growth factor-b binding protein (Ltbp)-3 null mice displaying growth retardation with multiple skeletal anomalies, defective thymus seems to be attributable to an involution rather than congenital hypoplasia because the mice show elevated corticosterone levels in the serum [3]. ...
... Thymic atrophy , accompanied with lymphopenia, is caused by the enhancement of BMP-4 signaling [8]. In mice deficient for VAMP8/endobrevin, which is associated with soluble-N-ethylmaleimide-sensitive factor attachment receptor (SNARE), the organization of the thymic epithelium and T cell development were severely affected in the mice [6] . Common pathological alterations in the thymuses of these knockout mice and pet/pet rats are reduced proliferation and increased apoptosis in their thymocytes. ...
The petit rat (pet/pet) is a new semi-lethal dwarf mutant with anomalies in the thymus and testes, defects inherited as a single autosomal recessive trait. At birth, these pet/pet rats show low birth weight and extremely small thymuses; at 140 days of age, their thymuses show abnormal involution. In the present study, we examined early postnatal development of hypoplastic pet/pet thymuses. In addition to being hypoplastic at birth, pet/pet thymus growth was almost completely impaired during the early postnatal period. As shown by cellular incorporation of BrdU, the mitotic activity was lower in pet/pet than in normal thymuses, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that apoptosis occurred more often in pet/pet than in normal thymus cells during the first few days after birth. These results indicate that postnatal development of the hypoplastic pet/pet thymus is defective due to the reduced proliferation and increased apoptosis of thymic cells.
... Recently, Kanwar et al. [15] described increased T-cell apoptosis in VAMP8-deficient mice. However, when we performed proliferation assay on mature CD8 1 T cells, we found that there is no difference in the proliferation rate between the WT and KO cells (data not shown). ...
... However, when we performed proliferation assay on mature CD8 1 T cells, we found that there is no difference in the proliferation rate between the WT and KO cells (data not shown). The observation described in the study [15] may be attributed to developmental stage difference between the KO and control mice since the thymus is overwhelmed with immature T cells and undergoes apoptosis 5-6 wk after birth. ...
CTL clear virus-infected cells and tumorigenic cells by releasing potent cytotoxic enzymes stored in preformed lytic granules. The exocytosis process includes polarization of lytic granules toward the immunological synapse, tethering of lytic granules to the plasma membrane and finally fusion of lytic granules with the plasma membrane to release cytotoxic enzymes. Although much is known about the molecular machineries necessary for the earlier steps in lytic granule exocytosis, the molecular machinery governing the final step in the fusion process has not been identified. Here, we show using control and VAMP8 KO mice that VAMP8 is localized to the CTL lytic granules. While the immunological synapse and granule polarization appears normal in both VAMP8 KO and control CTL, CTL-mediated killing was reduced for the Vamp8(-/-) CTL. Analysis of lytic enzyme secretion demonstrated that granzyme A and granzyme B secretion is significantly compromised in VAMP8(-/-) CTL, while the levels of the lytic enzymes in the cells are unaffected. Our results clearly show that VAMP8 is one of the v-SNARE that regulate the lytic ability of CTL by influencing the ability of the lytic granules to fuse with the plasma membrane and release its contents.
... Compared with control cells, after mDpy-30 knockdown, a pool of STX10 and VAMP3 also became enriched near cell protrusions (Fig. 4 A), where they exhibited a partial colocalization with internalized CD8-CIMPR (Fig. 4 B). However, VAMP4 and VAMP8, two other SNARE proteins along the TGN–PM–endosome loop (Cocucci et al., 2008; Kanwar et al., 2008), displayed little or only modest enrichment and little to no colocalization with CD8-CIMPR within this region (unpublished data). ...
Histone lysine methyltransferase complexes are essential for chromatin organization and gene regulation. Whether any of this machinery functions in membrane traffic is unknown. In this study, we report that mammal Dpy-30 (mDpy-30), a subunit of several histone H3 lysine 4 (H3K4) methyltransferase (H3K4MT) complexes, resides in the nucleus and at the trans-Golgi network (TGN). The TGN targeting of mDpy-30 is mediated by BIG1, a TGN-localized guanine nucleotide exchange factor for adenosine diphosphate ribosylation factor GTPases. Altering mDpy-30 levels changes the distribution of cation-independent mannose 6-phosphate receptor (CIMPR) without affecting that of TGN46 or transferrin receptor. Our experiments also indicate that mDpy-30 functions in the endosome to TGN transport of CIMPR and that its knockdown results in the enrichment of internalized CIMPR and recycling endosomes near cell protrusions. Much like mDpy-30 depletion, the knockdown of Ash2L or RbBP5, two other H3K4MT subunits, leads to a similar redistribution of CIMPR. Collectively, these results suggest that mDpy-30 and probably H3K4MT play a role in the endosomal transport of specific cargo proteins.
Parkinson's disease (PD) is the second most common neurodegenerative condition and intracellular deposition of Lewy bodies in the substantia nigra (SN), which can cause dopaminergic neuronal death, is the hallmark of this syndrome. α-synuclein (syn) is a small protein expressed mainly in neurons but can also be found in a number of tissues. It can be present as a soluble monomer under normal physiological conditions, but can be toxic in its oligomeric or fibrillary forms. Most of the available literature has focused on the effects of α-syn pathology in the mechanisms leading to PD. However, the normal functions of α-syn still remain to be fully elucidated. Notably, α-syn in the hematopoietic system seems to mediate important functions as indicated by anemia and incomplete cell maturation when this protein is absent. This review will summarize basic genetic and structural findings, and critical information that suggests an essential role of α-syn in the development and activation of the hematopoietic system and immunity.
