Hyun-Jeong Cho

DYRK1A-mediated hyperphosphorylation of Tau. A functional link between Down syndrome and Alzheimer disease.

Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of AD.

Dual‐specificity tyrosine(Y)‐phosphorylation regulated kinase 1A‐mediated phosphorylation of amyloid precursor protein: evidence for a functional link between Down syndrome and Alzheimer’s disease

Most individuals with Down Syndrome (DS) show an early‐onset of Alzheimer’s disease (AD), which potentially results from the presence of an extra copy of a segment of chromosome 21. Located on chromosome 21 are the genes that encode β‐amyloid (Aβ) precursor protein (APP ), a key protein involved in the pathogenesis of AD, and dual‐specificity tyrosine(Y)‐phosphorylation regulated kinase 1A (DYRK1A ), a proline‐directed protein kinase that plays a critical role in neurodevelopment. Here, we describe a potential mechanism for the regulation of AD pathology in DS brains by DYRK1A‐mediated phosphorylation of APP. We show that APP is phosphorylated at Thr668 by DYRK1A in vitro and in mammalian cells. The amounts of phospho‐APP and Aβ are increased in the brains of transgenic mice that over‐express the human DYRK1A protein. Furthermore, we show that the amounts of phospho‐APP as well as those of APP and DYRK1A are elevated in human DS brains. Taken together, these results reveal a potential regulatory link between APP and DYRK1A in DS brains, and suggest that the over‐expression of DYRK1A in DS may play a role in accelerating AD pathogenesis through phosphorylation of APP.

A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations.

Platelets, though anucleated, possess several transcription factors, including NF-kappaB, that exert non-genomic functions regulating platelet activation. Since platelets have not only been recognized as central players of homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and inflammation, we examined rat platelet NF-kappaB expression and evaluated the effects of anti-inflammatory drug BAY 11-7082, an inhibitor of NF-kappaB activation, in platelet physiology. Western blotting revealed that rat platelets express NF-kappaB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet aggregation. ATP release, TXB(2) formation, P-selectin expression, and intercellular Ca(2+) concentration activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA) inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the drug is considered as anti-atherothrombosis, and anti-inflammatory therapy.

Total Saponin from Korean Red Ginseng Inhibits Thromboxane A 2 Production Associated Microsomal Enzyme Activity in Platelets

Ginseng, the root of Panax ginseng Meyer, has been used frequently in traditional oriental medicine and is popular globally. Ginsenosides, which are the saponins in ginseng, are the major components having pharmacological and biological activities, including anti-diabetic and anti-tumor activities. In this study, we investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-produced thromboxane A2 (TXA2), an aggregating thrombogenic molecule, and its associated microsomal enzymes cyclooxygenase (COX)-1 and TXA2 synthase (TXAS). Thrombin (0.5 U/mL) increased TXA2 production up to 169 ng/108 platelets as compared with control (0.2 ng/108 platelets). However, TSKRG inhibited potently TXA2 production to the control level in a dose-dependent manner, which was associated with the strong inhibition of COX-1 and TXAS activities in platelet microsomes having cytochrome c reductase activity. The results demonstrate TSKRG is a beneficial traditional oriental medicine in platelet-mediated thrombotic diseases via suppression of COX-1 and TXAS to inhibit production of TXA2.

Inhibitory effects of total saponin from Korean red ginseng via vasodilator-stimulated phosphoprotein-Ser 157 phosphorylation on thrombin-induced platelet aggregation

In this study, we have investigated the effects of total saponin from Korean red ginseng (TSKRG) on thrombin-induced platelet aggregation. TSKRG dose-dependently inhibited thrombin-induced platelet aggregation with IC50 value of about 81.1 μg/mL. In addition, TSKRG dose-dependently decreased thrombin-elevated the level of cytosolic-free Ca2+ ([Ca2+]i), one of aggregation-inducing molecules. Of two Ca2+-antagonistic cyclic nucleotides as aggregation-inhibiting molecules, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), TSKRG significantly dose-dependently elevated intracellular level of cAMP, but not cGMP. In addition, TSKRG dose-dependently inhibited thrombin-elevated adenosine triphosphate (ATP) release from platelets. These results suggest that the suppression of [Ca2+]i elevation, and of ATP release by TSKRG are associated with upregulation of cAMP. TSKRG elevated the phosphorylation of vasodilator-stimulated phosphoprotein (VASP)-Ser157, a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser239, a cGMPdependent protein kinase substrate, in thrombin-activated platelets. We demonstrate that TSKRG involves in increase of cAMP level and subsequent elevation of VASP-Ser157 phosphorylation through A-kinase activation to inhibit [Ca2+]i mobilization and ATP release in thrombin-induced platelet aggregation. These results strongly indicate that TSKRG is a beneficial herbal substance elevating cAMP level in thrombin-platelet interaction, which may result in preventing of platelet aggregation-mediated thrombotic diseases.

Inhibition of platelet aggregation by chlorogenic acid via cAMP and cGMP-dependent manner.

In this study, we investigated the effect of chlorogenic acid, a phenolic acid, on collagen (10 &mgr;g/ml)-stimulated platelet aggregation. Chlorogenic acid dose-dependently inhibited collagen-induced platelet aggregation, and suppressed the production of thromboxane A2 (TXA2), an intracellular Ca2+-agonist as an aggregation-inducing autacoidal molecule, which was associated with the strong inhibition of cyclooxygenase (COX)-1 in platelet microsomes having cytochrome c reductase activity. In addition, chlorogenic acid increased significantly the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), intracellular Ca2+-antagonists as aggregation-inhibiting molecules. These results suggest that chlorogenic acid has antiplatelet activity through the reduction of TXA2 and the increase of cAMP and cGMP levels. Therefore, our data demonstrate that chlorogenic acid is a potent inhibitor of collagen-stimulated platelet aggregation, and may be a crucial tool for a negative regulator during platelet activation in thrombotic diseases.

Vasodilator-stimulated phosphoprotein-phosphorylation by ginsenoside Ro inhibits fibrinogen binding to αIIb/β3 in thrombin-induced human platelets

Background Glycoprotein IIb/IIIa (αIIb/β3) is involved in platelet adhesion, and triggers a series of intracellular signaling cascades, leading to platelet shape change, granule secretion, and clot retraction. In this study, we evaluated the effect of ginsenoside Ro (G-Ro) on the binding of fibrinogen to αIIb/β3. Methods We investigated the effect of G-Ro on regulation of signaling molecules affecting the binding of fibrinogen to αIIb/β3, and its final reaction, clot retraction. Results We found that G-Ro dose-dependently inhibited thrombin-induced platelet aggregation and attenuated the binding of fibrinogen to αIIb/β3 by phosphorylating cyclic adenosine monophosphate (cAMP)-dependently vasodilator-stimulated phosphoprotein (VASP; Ser157). In addition, G-Ro strongly abrogated the clot retraction reflecting the intensification of thrombus. Conclusion We demonstrate that G-Ro is a beneficial novel compound inhibiting αIIb/β3-mediated fibrinogen binding, and may prevent platelet aggregation-mediated thrombotic disease.

Cordycepin-Enriched WIB801C from Cordyceps militaris Inhibits Collagen-Induced (Ca 2+ ) i Mobilization via cAMP-Dependent Phosphorylation of Inositol 1, 4, 5-Trisphosphate Receptor in Human Platelets

In this study, we prepared cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha, and investigated the effect of CE-WIB801C on collagen-induced human platelet aggregation. CE-WIB801C dose-dependently inhibited collagen-induced platelet aggregation, and its IC50 value was 175 μg/ml. CE-WIB801C increased cAMP level more than cGMP level, but inhibited collagen-elevated [Ca2+]i mobilization and thromboxane A2 (TXA2) production. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased the CE-WIB801C-downregulated [Ca2+]i level in a dose dependent manner, and strongly inhibited CE-WIB801C-induced inositol 1, 4, 5-trisphosphate receptor (IP3R) phosphorylation. These results suggest that the inhibition of [Ca2+]i mobilization by CE-WIB801C is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CE-WIB801C suppressed TXA2 production, but did not inhibit the activities of cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS). These results suggest that the inhibition of TXA2 production by WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. In this study, we demonstrate that CE-WIB801C with cAMP-dependent Ca2+-antagonistic antiplatelet effects may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

Inhibitory effects of oligopeptides from hen egg white on both human platelet aggregation and blood coagulation

Egg white proteins have many biological functions and substantial nutritional benefits when used as a food source; however, they also contain allergens such as ovalbumin, ovomucoid, and ovotransferrin. We prepared oligopeptides without allergens from hen egg whites via the use of several proteases, and assessed their effects on platelet aggregation and blood coagulation, known to both of which are known to be major risk factors in thrombogenesis. Egg white oligopeptides (EWOP) inhibited collagen-induced human platelet aggregation in a dose-dependent manner. Additionally, we attempted to determine whether cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), aggregation-inhibiting intracellular molecules, regulate EWOP-inhibited platelet aggregation. EWOP caused an increase in cAMP levels, but did not affect cGMP levels, which suggests that the anti-platelet activity of EWOP operates in a cAMP-dependent manner, rather than via a cGMP-dependent process, in collagen-induced platelet aggregation. In addition, EWOP induced a significantly prolonged prothrombin time (PT) as compared with the controls. These data show that EWOP inhibits the conversion of fibrinogen to fibrin in a plasmatic atmosphere on an extrinsic pathway. Accordingly, these findings suggest that EWOP may be an excellent candidate as a crucial inhibitor of platelet activation, and its anti-platelet effects appear to involve the inhibition of both platelet aggregation and blood coagulation within the cardiovascular system.

Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets

Background Intracellular Ca2+([Ca2+]i) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of [Ca2+]i mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the Ca2+-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). Methods We investigated the Ca2+-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I (IP3RI) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. Results The inhibition of [Ca2+]i mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-Br-cAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) (Thr197) by KRG-TS. The phosphorylation of IP3RI (Ser1756) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-Br-cGMPS. These results suggest that the inhibitory effect of [Ca2+]i mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. Conclusion These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits [Ca2+]i mobilization in thrombin–platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.