SNAREs are undoubtedly one of the core elements of synaptic transmission. Contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the ...level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations in cultured mouse hippocampal neurons: (1) elongation of STX1A's JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; (2) charge reversal mutations in STX1A's JMD; and (3) palmitoylation deficiency mutations in STX1A's TMD. We found that both JMD elongations and charge reversal mutations have position-dependent differential effects on Ca
-evoked and spontaneous neurotransmitter release. Importantly, we show that STX1A's JMD regulates the palmitoylation of STX1A's TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1A's JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.
Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex ...through two major binding modes: through STX1's N-peptide and through STX1's closed conformation driven by its H
- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, H
-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the H
-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca
-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.
Synaptotagmin-1 (SYT1) is a synaptic vesicle resident protein that interacts via its C2 domain with anionic lipids from the plasma membrane in a calcium-dependent manner to efficiently trigger rapid ...neurotransmitter (NT) release. In addition, SYT1 acts as a negative regulator of spontaneous NT release and regulates synaptic vesicle (SV) priming. How these functions relate to each other mechanistically and what role other synaptotagmin (SYT) isoforms play in supporting and complementing the role of SYT1 is still under intensive investigation. In this work, we analyzed three putative functions of SYT1 in exocytosis by systematically varying its expression in autaptic hippocampal glutamatergic neurons from mice of either sex. We find that regulation of release probability is most sensitive to variation of expression levels, whereas its impact on vesicle priming is least sensitive. Also, loss of SYT1 phenotypes on spontaneous release and vesicle priming is compensated in less mature synaptic cultures by redundant support from SYT7. Overall, our data help in resolving some controversies in SYT1 functions in exocytosis and in our understanding of how SYT1 contributes to the pathophysiology underlying SYT1-related human neurologic disorders.
Our work clarifies the functions of SYT1 protein in synaptic vesicle priming and spontaneous and calcium-evoked neurotransmitter release and analyzes whether these occur at different stages of synaptic responses by examining their relative sensitivity to protein concentration at the synaptic terminal. We demonstrate that these synaptic functions are unequally sensitive to both protein levels and neuronal stage, indicating that they operate under distinct molecular mechanisms. Furthermore, we analyze how these functions are modulated by another synaptotagmin isoform expression. We show that to understand the phenotype displayed by SYT1 knock-out neurons (
) is necessary to consider the interplay between SYT1 and SYT7 molecules at the presynaptic terminal.
Background: Previous articles have identified the farnesoid X receptor (FXR) as an integral part in the formation of coated platelets. Coated platelets are preactivated platelets featuring ...degranulation, increased fibrinogen binding, and increased serine protease activity leading to fibrin generation. Furthermore, phosphatidylserine exposure is increased and integrin α2bβIII is inhibited - leading to a prothrombotic phenotype despite decreased platelet aggregation. We hypothesize that bile acids, as natural ligands of FXR, lead to a change of platelet phenotype and therefore play a pivotal role in the formation of coated platelets, especially in presence of cholestasis.
Methods: Based on previous findings, we incubated human washed platelets of healthy adult volunteers with the synthetic FXR ligand GW4064 in various concentrations (0, 10, 20, 50, 100µM) and used flow cytometry to detect a shift in p-selectin expression, PAC-1 binding and annexin-V-binding. Moreover, we used different concentrations (0, 100, 200, 400, 600µM) of three bile acids (ursodeoxycholic acid, UDCA; chenodeoxycholic acid, CDCA; glycochenodeoxycholic acid, GCDCA) to see if natural FXR ligands induce an effect on the platelet phenotype.
Results: We observed a dose dependent shift in annexin-V-binding when treating washed platelets with GW4064 as well as CDCA and GCDCA. Similarly, GW4064 led to increased p-selectin expression while increased PAC-1-binding was only detected at the highest concentration. In contrast, CDCA and GCDCA showed merely slight changes in p-selectin expression whereas PAC-1-binding seemed to be unaffected. However, none of these effects were seen when using UDCA.
Conclusion: We conclude that pretreatment of washed platelets with CDCA and GCDCA initiate a dose-dependent shift towards a prothrombotic platelet phenotype. Therefore, we assume that increased levels of certain bile acids drive thrombosis in patients with cholestatic liver injury. Furthermore, a recent mouse model study suggested that platelet derived growth factor β (PDGFβ), a component of α-granula, drives liver fibrosis. Hence, in addition to their prothrombotic effects, coated platelets might exacerbate liver fibrosis.
No relevant conflicts of interest to declare.
Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma ...and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms.
The potential of various bile acids to induce procoagulant tissue factor (TF) activity in viable HepG2 cells and primary human hepatocytes was investigated. Increased TF activity correlated with the molecules' ability to enter the cells and activate the farnesoid X receptor (FXR) suggesting a crucial role of FXR in bile acid-mediated TF activity within the liver parenchyma.
Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma ...and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms. The potential of various bile acids to induce procoagulant tissue factor (TF) activity in viable HepG2 cells and primary human hepatocytes was investigated. Increased TF activity correlated with the molecules' ability to enter the cells and activate the farnesoid X receptor (FXR) suggesting a crucial role of FXR in bile acid-mediated TF activity within the liver parenchyma.
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