Background:The ATP requirements of synaptic vesicle release are poorly understood. Results: Mitochondrially derived ATP supports the function of boutons with and without mitochondria. Respiratory dysfunction selectively blocks the reinternalization of synaptic vesicles. Conclusion: ATP diffuses rapidly in axons to support synaptic vesicle recycling. Mitochondrial dysfunction decreases synaptic energy and impairs function. Significance: Understanding energy requirements will help determine how energy failure contributes to neurodegeneration.

Efficient coupling of cellular energy production to metabolic demand is crucial to maintain organismal homeostasis. Here, we report that the mitochondrial Sirtuin Sirt4 regulates mitochondrial ATP homeostasis. We find that Sirt4 affects mitochondrial uncoupling via the adenine nucleotide translocator 2 (ANT2). Loss of Sirt4 expression leads to decreased cellular ATP levels in vitro and in vivo while Sirt4 overexpression is associated with increased ATP levels. Further, we provide evidence that lack of Sirt4 activates a retrograde signaling response from the mitochondria to the nucleus that includes AMPK, PGC1α, key regulators of β-oxidation such as Acetyl-CoA carboxylase, and components of the mitochondrial respiratory machinery. This study highlights the ability of Sirt4 to regulate ATP levels via ANT2 and a feedback loop involving AMPK.

No abstract

Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.

Lin CC, Lee IT, Wu WL, Lin WN, Yang CM. Adenosine triphosphate regulates NADPH oxidase activity leading to hydrogen peroxide production and COX-2/PGE2 expression in A549 cells. Am J Physiol Lung Cell Mol Physiol 303: L401-L412, 2012. First published July 6, 2012; doi:10.1152/ajplung.00090.2012.-Non-small cell lung carcinoma (NSCLC) accounts for most of all lung cancers, which is the leading cause of mortality in human beings. High level of cyclooxygenase-2 (COX-2) is one of the features of NSCLC and related to the low survival rate of NSCLC. However, whether extracellular nucleotides releasing from stressed resident tissues contributes to the expression of COX-2 remains unclear. Here, we showed that stimulation of A549 cells by adenosine 5=-O-(3-thiotriphosphate) (ATP␥S) led to an increase in COX-2 gene expression and prostaglandin E2 (PGE2) synthesis, revealed by Western blotting, RT-PCR, promoter assay, and enzyme-linked immunosorbent assay. In addition, ATP␥S induced intracellular reactive oxygen species (ROS) generation through the activation of NADPH oxidase. The increase of ROS level resulted in activation of the c-Src/epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ nuclear factor (NF)-B cascade. We also found that activated Akt was translocated into the nucleus and recruited with NF-B and p300 to form a complex. Thus, activation of p300 modulated the acetylation of histone H4 via the NADPH oxidase/c-Src/EGFR/PI3K/Akt/NF-B cascade stimulated by ATP␥S. Our results are the first to show a novel role of NADPH oxidase-dependent Akt/p65/p300 complex formation that plays a key role in regulating COX-2/PGE2 expression in ATP␥S-treated A549 cells. Taken together, we demonstrated that ATP␥S stimulated activation of NADPH oxidase, resulting in generation of ROS, which then activated the downstream c-Src/EGFR/PI3K/Akt/ NF-B/p300 cascade to regulate the expression of COX-2 and synthesis of PGE2 in A549 cells. Understanding the regulation of COX-2 expression and PGE 2 release by ATP␥S on A549 cells may provide potential therapeutic targets of NSCLC. inflammation; signaling transductions; oxidative stress; cyclooxygenase-2; human NON-SMALL CELL LUNG CARCINOMA (NSCLC) is the most common one of various types of lung cancers. During the processes of lung carcinogenesis, inflammation has been postulated to play a key role in these responses. It is found that there are constitutive high levels of inflammatory proteins, cyclooxygenase-2 (COX-2), and expression in human NSCLC compared with normal adjacent lung tissue (39). Despite various genetic alterations in lung cancer, the elevated expression of COX-2 is believed to be the central factor of cancer invasion, metastasis, angiogenesis, and decreased host immunity (27).Clinical statistics reveal that the extent of COX-2 expression is associated with a lower overall survival rate and a diminished disease-free survival rate (22).COX is the rate-limiting enzyme for the production of eicosanoid prostaglandins and thromboxanes ...

Auto-reactive B lymphocytes and its abnormal CD40 signaling play important roles in the pathogenesis of systemic lupus erythematosus (SLE). In this study, we analyzed CD40 expression and CD40/CD154 induced activation of NF-κB signaling pathway in B cells from SLE patients. B cells from healthy volunteers and tonsilar B cells from chronic tonsillitis were used as negative and positive controls. Results showed CD40-induced NF-κB signaling was constitutively activated in B cells from active lupus patients, including decreased CD40 in raft portion, increased phosphorylation and degradation of IκBα, phosphorylation of P65, as well as increased nuclear translocation of P65, P50, c-Rel, which could be blocked by anti-CD154. CD154 stimulation could induce further phosphorylation and degradation of IκBα, as well as phosphorylation of P65 and nuclear translocation of P65. In addition, CD40-induced kinase activities in B cells from lupus patients mimicked that of tonsil B cells, in that IKKα/β were more activated compared to normal B cells. CD40-induced NF-κB activity was blocked by both IκB phosphorylation and proteosome degradation inhibitors in both lupus and normal B cells. All together, our findings revealed that canonical NF-κB signaling is constitutively activated in active lupus and is mediated by CD154/CD40. CD40 induced NF-κB activation is different in human lupus B lymphocytes compared with normal B cells.

Background /Aims: Accumulating evidence indicates that endothelial inflammation is one of the critical determinants in pathogenesis of atherosclerotic cardiovascular disease. Our previous studies had demonstrated that Vaccariae prevented high glucose or oxidative stress-triggered endothelial dysfunction in vitro. Very little is known about the potential effects of hypaphorine from Vaccariae seed on inflammatory response in endothelial cells. Methods: In the present study, we evaluated the anti-inflammatory effects of Vaccariae hypaphorine (VH) on lipopolysaccharide (LPS)-challenged endothelial EA.hy926 cells. The inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1) and vascular cellular adhesion molecule-1 (VCAM-1) were measured by real-time PCR (RT-PCR). The expressions of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), toll-like receptor 4 (TLR4), peroxisome proliferator-activated receptor γ (PPARγ) were detected by Western blotting or immunofluorescence. Results: We showed that LPS stimulated the expressions of TNF-α, IL-1β, MCP-1, VCAM-1 and TLR4, but attenuated the phosphorylation of AMPK and ACC as well as PPARγ protein levels, which were reversed by VH pretreatment. Moreover, we observed that LPS-upregulated TLR4 protein expressions were inhibited by PPARγ agonist pioglitazone, and the downregulated PPARγ expressions in response to LPS were partially restored by knockdown of TLR4. The negative regulation loop between TLR4 and PPARγ response to LPS was modulated by AMPK agonist AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) or A769662. Conclusions: Taken together, our results suggested that VH ameliorated LPS-induced inflammatory cytokines production in endothelial cells via inhibition of TLR4 and activation of PPARγ, dependent on AMPK signalling pathway.

Rationale:The Wolff–Parkinson–White syndrome (WPW) is a benign heart disease with accessory pathways, which can result in cardiac arrhythmias. The purpose of this case report is to introduce a rare case of sudden cardiac death (SCD) with a mild myocardial bridge and a history of WPW.Patient concerns:A 25-year-old man with known WPW syndrome died at night while sleeping.Diagnoses:Diagnosis of WPW syndrome is based on typical electrocardiogram findings with a documented dysrhythmia before the victim's death.Interventions:At autopsy, no traumatic injury or common poisons were found, only a slight myocardial bridge was detected. We performed whole exome sequencing and identified several genetic variations related to SCD.Outcomes:We considered that the cause of death in this case was SCD in which arrhythmia might play an important role.Lessons:This case highlights SCD can occur in WPW patients with mild or unrecognized structural abnormality. Postmortem genetic examination can assist the diagnosis of sudden cardiac death, especially when no lethal structural abnormality is found in the decedent.