Impairment of PARK14-dependent Ca(2+) signalling is a novel determinant of Parkinson's disease.

The etiology of idiopathic Parkinson's disease (idPD) remains enigmatic despite recent successes in identification of genes (PARKs) that underlie familial PD. To find new keys to this incurable neurodegenerative disorder we focused on the poorly understood PARK14 disease locus (Pla2g6 gene) and the store-operated Ca(2+) signalling pathway. Analysis of the cells from idPD patients reveals a significant deficiency in store-operated PLA2g6-dependent Ca(2+) signalling, which we can mimic in a novel B6.Cg-Pla2g6(ΔEx2-VB) (PLA2g6 ex2(KO)) mouse model. Here we demonstrate that genetic or molecular impairment of PLA2g6-dependent Ca(2+) signalling is a trigger for autophagic dysfunction, progressive loss of dopaminergic (DA) neurons in substantia nigra pars compacta and age-dependent L-DOPA-sensitive motor dysfunction. Discovery of this previously unknown sequence of pathological events, its association with idPD and our ability to mimic this pathology in a novel genetic mouse model opens new opportunities for finding a cure for this devastating neurodegenerative disease.

Role of molecular determinants of store-operated Ca(2+) entry (Orai1, phospholipase A2 group 6, and STIM1) in focal adhesion formation and cell migration.

BACKGROUND: Store-operated Ca(2+) entry is important for cell migration. RESULTS: This study presents characterization of localization and roles of Orai1, STIM1, and PLA2g6 in adhesion dynamics during cell migration. CONCLUSION: Orai1 and PLA2g6 are involved in adhesion formation at the front, whereas STIM1 participates in both adhesion formation and disassembly. SIGNIFICANCE: Results uncovered new parameters of Orai1, STIM1, and PLA2g6 involvement in cell migration. Store-operated Ca(2+) entry and its major determinants are known to be important for cell migration, but the mechanism of their involvement in this complex process is unknown. This study presents a detailed characterization of distinct roles of Orai1, STIM1, and PLA2g6 in focal adhesion (FA) formation and migration. Using HEK293 cells, we discovered that although molecular knockdown of Orai1, STIM1, or PLA2g6 resulted in a similar reduction in migration velocity, there were profound differences in their effects on number, localization, and lifetime of FAs. Knockdown of STIM1 caused an increase in lifetime and number of FAs, their redistribution toward lamellae region, and an increase in cell tail length. In contrast, the number of FAs in Orai1- or PLA2g6-deficient cells was significantly reduced, and FAs accumulated closer to the leading edge. Assembly rate and Vinculin phosphorylation of FAs was similarly reduced in Orai1, PLA2g6, or STIM1-deficient cells. Although Orai1 and PLA2g6 accumulated and co-localized at the leading edge, STIM1 distribution was more complex. We found STIM1 protrusions in lamellipodia, which co-localized with FAs, whereas major accumulation could be seen in central and retracting parts of the cell. Interestingly, knockdown of Orai1 and PLA2g6 produced similar and non-additive effect on migration, whereas knockdown of STIM1 simultaneously with either Orai1 or PLA2g6 produced additional inhibition. Together these data suggest that although Orai1, PLA2g6, and STIM1 play major roles in formation of new FAs at the leading edge, STIM1 may also be involved in Orai1- and PLA2g6-independent disassembly of FAs in the back of cells.

Modulation of Ca(2+) release through ryanodine receptors in vascular smooth muscle by protein kinase Calpha.

The role of protein kinase C (PKC) in Ca(2+) release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca(2+)](i) transients. Application of the PKC inhibitor calphostin C caused [Ca(2+)](i) transients which were not blocked by nifedipine or by removing extracellular Ca(2+) but were abolished after inhibition of the SR Ca(2+)-ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca(2+)](i) but no further [Ca(2+)](i) increase was seen with subsequent application of caffeine. Selective inhibition of PKCalpha with safingol blocked caffeine-induced [Ca(2+)](i) transients, but the PKCepsilon inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCalpha, caffeine-induced [Ca(2+)](i) transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCalpha in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCalpha indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca(2+)-dependent PKCalpha isoform.

Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity.

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.

Hypoxia influences CD40-CD40L mediated inflammation in endothelial and monocytic cells.

The interaction between CD40 and its ligand (CD40L) has been implicated in the pathogenesis of atherosclerosis and is recognized as a central event in the development of immuno-inflammatory processes. Our previous studies have shown that the CD40-CD40L interaction modulates platelet, neutrophil, and endothelial reactive oxygen species (ROS) generation. Hypoxia, known to be associated with tissue ischemia and inflammation, also influences the ROS production and changes the cellular redox state. However, the effect of hypoxia on CD40-CD40L mediated vascular inflammation is unknown. We have investigated whether hypoxia influences CD40-CD40L mediated vascular inflammatory responses, ROS production, and cellular interactions. We found that hypoxia significantly enhances the inflammatory effect of CD40L in both endothelial and monocytic cells (THP1). CD40-CD40L interaction in the presence of hypoxia induces ROS production, the synthesis of an inflammatory adhesive protein intercellular adhesion molecule 1 (ICAM1) and activates stress response proteins (p38 MAP kinase and HSP27), indicating that CD40L mediates the induction of oxidative stress in these cells. Importantly, we found that the effects of CD40L can be transmitted between HUVECs and monocytic THP1 cells through intercellular CD40-CD40L interaction and these processes are augmented under hypoxia. Together, these data indicate that under hypoxic conditions the CD40-CD40L interaction significantly influences adhesion molecule expression, stress generation, actin polymerization, and monocytic adhesion to endothelial cells in addition to changes in signaling. In summary, we show that hypoxia can alter CD40-CD40L mediated endothelial-monocyte interaction, playing a significant role in vascular inflammation and cellular adhesion processes.

Regulation of ROS signal transduction by NADPH oxidase 4 localization.

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

Adiponectin modulates inflammatory reactions via calreticulin receptor-dependent clearance of early apoptotic bodies.

Obesity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function. Adiponectin-deficient (APN-KO) mice were impaired in their ability to clear apoptotic thymocytes in response to dexamethasone treatment, and these animals displayed a reduced ability to clear early apoptotic cells that were injected into their intraperitoneal cavities. Conversely, adiponectin administration promoted the clearance of apoptotic cells by macrophages in both APN-KO and wild-type mice. Adiponectin overexpression also promoted apoptotic cell clearance and reduced features of autoimmunity in lpr mice whereas adiponectin deficiency in lpr mice led to a further reduction in apoptotic cell clearance, which was accompanied by exacerbated systemic inflammation. Adiponectin was capable of opsonizing apoptotic cells, and phagocytosis of cell corpses was mediated by the binding of adiponectin to calreticulin on the macrophage cell surface. We propose that adiponectin protects the organism from systemic inflammation by promoting the clearance of early apoptotic cells by macrophages through a receptor-dependent pathway involving calreticulin.

H(2)O(2): a mediator of esophagitis-induced damage to calcium-release mechanisms in cat lower esophageal sphincter.

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca(2+) stores. We therefore measured cytosolic Ca(2+) in response to a maximally effective dose of ACh in fura 2-AM-loaded lower esophageal sphincter (LES) circular muscle cells and examined the contribution of H(2)O(2) to the reduction in Ca(2+) signal. In normal cells, the ACh-induced Ca(2+) increase was the same in normal-Ca(2+) and Ca(2+)-free medium and was abolished by the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C inhibitor U-73122, confirming that the initial ACh-induced contraction depends on Ca(2+) release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced Ca(2+) increase in normal-Ca(2+) medium was significantly lower in esophagitis than in normal cells and was further reduced ( approximately 70%) when the cells were incubated in Ca(2+)-free medium. This reduction was partially reversed by the H(2)O(2) scavenger catalase. H(2)O(2) measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with H(2)O(2), the ACh-induced Ca(2+) increase was significantly reduced in normal-Ca(2+) and Ca(2+)-free medium and was similar to that observed in animals with esophagitis. The initial ACh-induced contraction was also reduced in normal cells incubated with H(2)O(2). H(2)O(2), when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca(2+) signal in normal cells. H(2)O(2)-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, H(2)O(2) reduced TG-induced contraction, suggesting that TG and H(2)O(2) may operate through similar mechanisms. Ca(2+)-ATPase activity measurement indicates that H(2)O(2) and TG reduced Ca(2+)-ATPase activity, confirming similarity of mechanism of action. We conclude that H(2)O(2) may be at least partly responsible for impairment of Ca(2+) release in acute experimental esophagitis by inhibiting Ca(2+) uptake and refilling Ca(2+) stores.

Hydrogen peroxide contributes to motor dysfunction in ulcerative colitis.

Ulcerative colitis (UC) affects colonic motor function, but the mechanism responsible for this motor dysfunction is not well understood. We have shown that neurokinin A (NKA) may be an endogenous neurotransmitter mediating contraction of human sigmoid colonic circular muscle (HSCCM). To elucidate factors responsible for UC motor dysfunction, we examined the role of hydrogen peroxide (H(2)O(2)) in the decrease of NKA-induced response of HSCCM. As previously demonstrated, NKA-induced contraction or Ca(2+) increase of normal muscle cells is mediated by release of Ca(2+) from intracellular stores, because it was not affected by incubation in Ca(2+)-free medium (CFM) containing 200 microM BAPTA. In UC, however, CFM reduced both cell contraction and NKA-induced Ca(2+) increase, suggesting reduced Ca(2+) release from intracellular stores. In normal Ca(2+) medium, NKA and KCl caused normal Ca(2+) signal in UC cells but reduced cell shortening. The decreased Ca(2+) signal and contraction in response to NKA or thapsigargin were partly recovered in the presence of H(2)O(2) scavenger catalase, suggesting involvement of H(2)O(2) in UC-induced dysmotility. H(2)O(2) levels were higher in UC than in normal HSCCM, and enzymatically isolated UC muscle cells contained much higher levels of H(2)O(2) than normal cells, which were significantly reduced by catalase. H(2)O(2) treatment of normal cells in CFM reproduced the reduction of NKA-induced Ca(2+) release observed in UC cells. In addition, H(2)O(2) caused a measurable, direct release of Ca(2+) from intracellular stores. We conclude that H(2)O(2) may contribute to reduction of NKA-induced Ca(2+) release from intracellular Ca(2+) stores in UC and contribute to the observed colonic motor dysfunction.