Caused by the presence of an extra copy of human chromosome 21 (trisomy 21), Down syndrome (DS) is the most common genetic disorder with an incidence of one in 800 live births. DS patients suffer various symptoms, including mental retardation and an early-onset of Alzheimer's disease (AD). The brains of both DS and AD patients show increased amounts of b-amyloid (Ab), which leads to the formation of amyloid plaques, a hallmark of AD pathogenesis (Mann and Esiri 1989). Although the cause of an early-onset AD in DS patients is not clearly understood (Wisniewski et al. 1985), one potential mechanism is overexpression of the gene for b-amyloid precursor protein (APP) located on chromosome 21. Three copies of the APP gene are necessary for DS-affected individuals to develop AD pathology (Prasher et al. 1998). However, this genetic background does not sufficiently account for the full spectrum of pathologies seen in AD patients; over-expres- Abbreviations used: aa, amino acids; AD, Alzheimer's disease; APP, amyloid precursor protein; APPct, C-terminal fragment of APP; Ab, b-amyloid; BACE1, b-secretase APP Cleaving Enzyme 1; DS, Down syndrome; DYRK1A, dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A; GFP, green fluorescent protein; GST, glutathione S-transferase; NGF, nerve growth factor; p-AICD, phospho-APP intracellular domain; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TG, transgenic; WT, wild-type; YF, Y321F kinase-inactive mutant. AbstractMost individuals with Down Syndrome (DS) show an earlyonset 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 b-amyloid (Ab) 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 Ab 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 overexpression of DYRK1A in DS may play a role in accelerating AD pathogenesis through phosphorylation of APP.

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. Down syndrome (DS)3 is the most common genetic disorder with a frequency of 1 in 800 live births, and it is caused by the presence of an extra copy of whole or part of human chromosome 21 (1, 2). DS patients suffer various symptoms, including congenital heart defects, immune and endocrine system defects, mental retardation, and early onset of Alzheimer disease (AD) (3). Both DS and AD patients have pathological hallmarks, amyloid plaques and neurofibrillary tangles (NFTs) that are insoluble deposits made of proteins called ␤-amyloid (A␤) and hyperphosphorylated Tau, respectively (4 -6). Although an early onset AD in DS patients is not clearly understood, one potential mechanism is the presence of three chromosomal copies of ␤-amyloid precursor protein (APP) gene. However, the APP overexpression alone in mice does not show the endosome abnormalities observed in AD-like pathology (7), implying the necessity of additional genes on the chromosome 21 for a full spectrum of AD pathologies.NFTs found in AD are composed of paired helical filaments (PHFs), which are mainly composed of hyperphosphorylated Tau protein (8). To date, more than 30 phosphorylation sites and 7-10 mol of phosphates per mol of Tau have been observed in PHF-Tau (9, 10). Although Tau protein is phosphorylated in vitro by numerous kinases, it is unclear how many kinases actually phosphorylate Tau in vivo. Currently, only glycogen synthase kinase 3␤ (GSK3␤), cyclin-dependent kinas...

The dual-specificity tyrosine(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) gene is located on human chromosome 21 and encodes a proline-directed protein kinase that might be responsible for mental retardation and early onset of Alzheimer's disease (AD) in Down syndrome (DS) patients. Presenilin 1 (PS1) is a key component of the c-secretase complex in the generation of b-amyloid (Ab), an important trigger protein in the pathogenesis of AD. Increased Dyrk1A expression has been reported in human AD and DS brains. We previously showed that Dyrk1A increased Ab production in mammalian cells and transgenic mice that over-express Dyrk1A. In this study, we describe a potential mechanism by which Ab is increased in Dyrk1A-over-expressing DS and AD brains. First, we show that PS1 is phosphorylated by the Dyrk1A at Thr 354 and that this phosphorylation increases c-secretase activity. Then, using transgenic mice that over-express human Dyrk1A, we demonstrate that phospho-Thr354-PS1 (pT354-PS1) expression is enhanced when Dyrk1A level is increased. We also show that pT354-PS1 is more stable than the unphosphorylated form of PS1. These results reveal a potential regulatory link between Dyrk1A and PS1 in the Ab pathway of DS and AD brains, suggesting that up-regulated Dyrk1A may accelerate AD pathogenesis through PS1 phosphorylation.

We compared the effects of genistein and daidzein on the expression of chemokines, cell adhesion molecules (CAMs), and endothelial nitric oxide synthase (eNOS) in tumor necrosis factor (TNF)-α-stimulated human umbilical vascular endothelial cells (HUVECs). TNF-α exposure significantly increased expression of monocyte chemoattractant protein (MCP)-1, vascular adhesion molecule (VCAM)-1, and intercellular adhesion molecule-1. Genistein significantly decreased MCP-1 and VCAM-1 production in a dose-dependent manner, whereas CAM expression was not significantly lowered by genistein treatment. However, daidzein slightly decreased MCP-1 production. The effects of genistein and daidzein on MCP-1 secretion coincided with mRNA expression. Pre-treatment with either genistein or daidzein elevated eNOS expression and nitric oxide production disturbed by TNF-α exposure. A low concentration of isoflavones significantly inhibited nuclear factor (NF)κB activation, whereas a high dose slightly ameliorated these inhibitive effects. These results suggest that genistein had a stronger effect on MCP-1 and eNOS expression than that of daidzein. Additionally, NFκB transactivation might be partially related to the down-regulation of these mRNAs in TNF-α-stimulated HUVECs.

This study characterized the efficacy of recombinant Cheonggukjang kinase (CGK) 3-5-rich fraction as a thrombolytic agent, which we overexpressed in Bacillus licheniformis ATCC10716, a strain normally lacking fibrinolytic activity. We found that CGK3-5 is a plasmin-like protease that directly degrades fibrin clots and does not activate plasminogen during fibrin clot lysis and platelet-rich clot lysis assays. We also confirmed antiplatelet and antithrombotic activity by CGK3-5-rich fraction both in vitro and in vivo. CGK3-5-rich fraction inhibited collagen-induced platelet aggregation in platelet-rich plasma in a concentration-dependent manner. The concentration of 1.5 mg/mL CGK3-5-rich fraction completely inhibited collagen-induced platelet aggregation. Furthermore, injection of CGK3-5-rich fraction into tail veins dose-dependently protected mice from death by pulmonary embolism induced by collagen and epinephrine. The survival rates were 30%, 70%, and 100%, respectively, with doses of 130 mg/kg, 260 mg/kg, and 520 mg/kg. These findings suggest that CGK3-5 holds promise as a treatment to mitigate the potentially effects of stroke and heart failure.