Citicoline ameliorates arsenic-induced hepatotoxicity and diabetes in mice by overexpression of VAMP2, PPAR-γ, As3MT, and SIRT3.

The use of arsenic in arsenic-based pesticides has been common in many countries in the past and today. There is considerable evidence linking arsenic exposure to hepatotoxicity and diabetes. Destructive phenomena such as hepatic oxidative stress and inflammation can interfere with glucose uptake and insulin function. In the present study, the antioxidant, anti-inflammatory, and molecular mechanism of citicoline against sodium arsenite-induced hepatotoxicity and glucose intolerance were investigated in mice. Citicoline improved glucose tolerance impaired by sodium arsenite. Citicoline increased the hepatic activity of catalase, superoxide dismutase, and glutathione peroxidase enzymes. Moreover, we found that citicoline prevents an increase in the levels of thiobarbituric acid reactive substances. Citicoline reduced levels of caspase 3, tumor necrosis factor-alpha, and interleukin 6 in sodium arsenite intoxicated groups. It was shown that citicoline increased the expression of arsenite methyltransferase, vesicle-associated membrane protein 2, peroxisome proliferator-activated receptor gamma, and sirtuin 3 to combat sodium arsenite toxicity. Citicoline reduced glucose intolerance, which was disrupted by sodium arsenite, by affecting the pancreatic and extra-pancreatic pathways involved in insulin production, secretion, and action. Based on our results, citicoline can be considered a modulating agent against arsenic-induced hepatotoxicity and hyperglycemia. Considering the relationship between arsenic exposure and the occurrence of side effects such as liver toxicity and diabetes, it is necessary to monitor and awareness of arsenic residues from sources such as drinking water.

Ferroportin-dependent ferroptosis induced by ellagic acid retards liver fibrosis by impairing the SNARE complexes formation.

Chronic liver injury causing liver fibrosis is a major cause of morbidity and mortality worldwide. Targeting the suppression of hepatic stellate cell (HSC) activation is recognized as an effective strategy for the treatment of liver fibrosis. Ellagic acid (EA), a natural polyphenol product isolated from fruits and vegetables, possesses many biological functions. Here, EA exerts its antifibrotic activity by inducing ferroptotic cell death of activated HSCs, which is accompanied by redox-active iron accumulation, lipid peroxidation, and GSH depletion in CCl4 mice and human LX-2 cells. The specific ferroptosis inhibitor ferrostatin-1 prevented EA-induced ferroptotic cell death. Mechanistically, EA impairs the formation of vesicle-associated membrane protein 2 (VAMP2)/syntaxin 4 and VAMP2/synaptosome-associated protein 23 complexes by suppressing VAMP2 expression by enhancing its degradation in a proteasome-dependent pathway. This leads to the impairment of ferroportin (FPN, an iron exporter) translocation and intracellular iron extrusion. Interestingly, VAMP2 overexpression inhibits the role of EA in blocking FPN translocation and increasing intracellular ferritin content (an iron storage marker). In contrast, VAMP2 knockdown shows a synergistic effect on EA-mediated ferroptotic events in both HSCs. Additionally, HSC-specific overexpression of VAMP2 impaired EA-induced HSC ferroptosis in mouse liver fibrosis, and HSC-specific VAMP2 knockdown increased the inhibitory effect of EA on fibrosis. Taken together, our data suggest that the natural product EA exerts its antifibrotic effects by inducing FPN-dependent ferroptosis of HSCs by disrupting the formation of SNARE complexes, and EA will hopefully serve as a prospective compound for liver fibrosis treatment.

Tetanus insensitive VAMP2 differentially restores synaptic and dense core vesicle fusion in tetanus neurotoxin treated neurons.

The SNARE proteins involved in the secretion of neuromodulators from dense core vesicles (DCVs) in mammalian neurons are still poorly characterized. Here we use tetanus neurotoxin (TeNT) light chain, which cleaves VAMP1, 2 and 3, to study DCV fusion in hippocampal neurons and compare the effects on DCV fusion to those on synaptic vesicle (SV) fusion. Both DCV and SV fusion were abolished upon TeNT expression. Expression of tetanus insensitive (TI)-VAMP2 restored SV fusion in the presence of TeNT, but not DCV fusion. Expression of TI-VAMP1 or TI-VAMP3 also failed to restore DCV fusion. Co-transport assays revealed that both TI-VAMP1 and TI-VAMP2 are targeted to DCVs and travel together with DCVs in neurons. Furthermore, expression of the TeNT-cleaved VAMP2 fragment or a protease defective TeNT in wild type neurons did not affect DCV fusion and therefore cannot explain the lack of rescue of DCV fusion by TI-VAMP2. Finally, to test if two different VAMPs might both be required in the DCV secretory pathway, Vamp1 null mutants were tested. However, VAMP1 deficiency did not reduce DCV fusion. In conclusion, TeNT treatment combined with TI-VAMP2 expression differentially affects the two main regulated secretory pathways: while SV fusion is normal, DCV fusion is absent.

Docking, not fusion, as the rate-limiting step in a SNARE-driven vesicle fusion assay.

In vitro vesicle fusion assays that monitor lipid mixing between t-SNARE and v-SNARE vesicles in bulk solution exhibit remarkably slow fusion on the nonphysiological timescale of tens of minutes to several hours. Here, single-vesicle, fluorescence resonance energy transfer-based assays cleanly separate docking and fusion steps for individual vesicle pairs containing full-length SNAREs. Docking is extremely inefficient and is the rate-limiting step. Of importance, the docking and fusion kinetics are comparable in the two assays (one with v-SNARE vesicles tethered to a surface and the other with v-SNARE vesicles free in solution). Addition of the V(C) peptide synaptobrevin-2 (syb(57-92)) increases the docking efficiency by a factor of ∼30, but docking remains rate-limiting. In the presence of V(C) peptide, the fusion step occurs on a timescale of ∼10 s. In previous experiments involving bulk fusion assays in which the addition of synaptotagmin/Ca(2+), Munc-18, or complexin accelerated the observed lipid-mixing rate, the enhancement may have arisen from the docking step rather than the fusion step.

The carboxy-terminal domain of complexin I stimulates liposome fusion.

Regulated exocytosis requires tight coupling of the membrane fusion machinery to a triggering signal and a fast response time. Complexins are part of this regulation and, together with synaptotagmins, control calcium-dependent exocytosis. Stimulatory and inhibitory functions have been reported for complexins. To test if complexins directly affect membrane fusion, we analyzed the 4 known mammalian complexin isoforms in a reconstituted fusion assay. In contrast to complexin III (CpxIII) and CpxIV, CpxI and CpxII stimulated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-pin assembly and membrane fusion. This stimulatory effect required a preincubation at low temperature and was specific for neuronal t-SNAREs. Stimulation of membrane fusion was lost when the carboxy-terminal domain of CpxI was deleted or serine 115, a putative phosphorylation site, was mutated. Transfer of the carboxy-terminal domain of CpxI to CpxIII resulted in a stimulatory CpxIII-I chimera. Thus, the carboxy-terminal domains of CpxI and CpxII promote the fusion of high-curvature liposomes.