Sul-Hee Chung

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.

Regulation of RCAN1 Protein Activity by Dyrk1A Protein-mediated Phosphorylation

Two genes on chromosome 21, namely dual specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) and regulator of calcineurin 1 (RCAN1), have been implicated in some of the phenotypic characteristics of Down syndrome, including the early onset of Alzheimer disease. Although a link between Dyrk1A and RCAN1 and the nuclear factor of activated T cells (NFAT) pathway has been reported, it remains unclear whether Dyrk1A directly interacts with RCAN1. In the present study, Dyrk1A is shown to directly interact with and phosphorylate RCAN1 at Ser112 and Thr192 residues. Dyrk1A-mediated phosphorylation of RCAN1 at Ser112 primes the protein for the GSK3β-mediated phosphorylation of Ser108. Phosphorylation of RCAN1 at Thr192 by Dyrk1A enhances the ability of RCAN1 to inhibit the phosphatase activity of calcineurin (Caln), leading to reduced NFAT transcriptional activity and enhanced Tau phosphorylation. These effects are mediated by the enhanced binding of RCAN1 to Caln and its extended half-life caused by Dyrk1A-mediated phosphorylation. Furthermore, an increased expression of phospho-Thr192-RCAN1 was observed in the brains of transgenic mice overexpressing the Dyrk1A protein. These results suggest a direct link between Dyrk1A and RCAN1 in the Caln-NFAT signaling and Tau hyperphosphorylation pathways, supporting the notion that the synergistic interaction between the chromosome 21 genes RCAN1 and Dyrk1A is associated with a variety of pathological features associated with DS.

Dyrk1A‐mediated phosphorylation of Presenilin 1: a functional link between Down syndrome and Alzheimer’s disease

J. Neurochem. (2010) 115, 574–584.

Phosphorylation and Inactivation of Glycogen Synthase Kinase 3β (GSK3β) by Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A)

Background: The regulatory mechanism of GSK3β activity is not yet fully understood. Results: Dyrk1A inactivates GSK3β by phosphorylation at Thr356, which may contribute to an obesity-resistant phenotype. Conclusion: Dyrk1A-mediated phosphorylation is an alternative pathway for GSK3β inactivation. Significance: Understanding the mechanism regulating GSK3β activity is crucial for developing new therapies against GSK3β-associated diseases, including obesity. Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr356 residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)356-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β.

Phosphorylation of Munc18‐1 by Dyrk1A regulates its interaction with Syntaxin 1 and X11α

J. Neurochem. (2012) 122, 1081–1091.

Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells

The mechanisms that underlie the altered neurotransmitter system in Alzheimers disease (AD) are not well understood. Amyloid precursor protein (APP) is a precursor protein for beta-amyloid, an important trigger protein in the pathogenesis of AD. Duplication of the APP gene as well as APP genes that contain certain mutations has been reported to be associated with familial AD (FAD), and a role of APP in neurotransmission has been suggested recently. This study examines the role of APP in exocytosis in PC12 cells using transfected human growth hormone (hGH) as a reporter for secretion. It was found that overexpression of APP or expression of the Swedish FAD mutation (APPsw) in PC12 cells significantly increased the basal secretion and constitutive secretion of hGH. Expression of an APP phosphorylation-deficient mutant decreased both basal and constitutive secretion relative to the APP wild-type, suggesting a role for APP-Thr668 phosphorylation in secretion in PC12 cells. Overexpression of X11alpha, a protein that stabilizes cellular APP, also increased the basal secretion of hGH but, contrary to APP, decreased the constitutive secretion of hGH, suggesting that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion of hGH may result from APP-X11alpha interaction. These results demonstrate an unknown role for APP in secretion, and suggest that elevated levels of APP or APP mutation in FAD brains contribute to the altered neurotransmitter pathology of AD through stimulation of basal and constitutive secretion.

Overexpression of BACE1 stimulates spontaneous basal secretion in PC12 cells

Although alterations in the function of the neurotransmitter system have been implicated in the pathology of Alzheimers disease (AD), the mechanisms that underlie this pathological change are not well understood. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is a key protease in the generation of beta-amyloid, an important trigger protein in the pathogenesis of AD. The expression and activity of BACE1 are increased in the brains of sporadic AD patients, and a role for BACE1 in neurotransmission has been suggested recently. This study examines whether BACE1 plays a role in regulated exocytosis in PC12 cells. Treatment of PC12 cells with a beta-secretase inhibitor reduced stimulus-dependent secretion of neurotransmitters, suggesting a potential role of BACE1 in regulated exocytosis. Using transfected human growth hormone as a reporter for a regulated secretory pathway in PC12 cells, we found that the transient overexpression of BACE1 increased basal secretion in the absence of a stimulus and reduced stimulus-dependent secretion in intact PC12 cells. In digitonin-permeabilized PC12 cells, an overexpression of BACE1 enhanced the Ca2+-independent and ATP-independent component of the secretory pathway. Furthermore, expression of the glycosylation-deficient mutant of BACE1, BACE1N354Q, led to an elevation of basal secretions over that by BACE1 wild-type, suggesting a role of BACE1 glycosylation in basal secretion. These results demonstrate an unknown role for BACE1 in secretion, and suggest that elevated levels of BACE1 in AD brains may contribute to the altered neurotransmitter pathology of AD through stimulation of spontaneous basal secretion under resting conditions.

Roscovitine increases intracellular calcium release and capacitative calcium entry in PC12 cells

Cyclin-dependent kinase 5 (Cdk5), which is activated by the non-cyclin regulator p35 or p39, is a proline-directed serine/threonine kinase that is implicated in many physiological and pathological processes. Here, we studied calcium signaling using the fluorescent cytosolic calcium indicator, Fura-4, in NGF-differentiated PC12 cells treated with roscovitine, a Cdk5 inhibitor. As compared to the control cells, the roscovitine-treated cells significantly potentiated intracellular calcium release by membrane depolarization (high K(+)) or through thapsigargin. In addition, roscovitine increased the magnitude of capacitative calcium entry (CCE), i.e., a calcium influx mechanism triggered by the depletion of intracellular calcium stores. Notably, roscovitine markedly slowed the rate of Ca(2+) removal from the plasma membrane. These results suggest that Cdk5 regulates intracellular calcium homeostasis and that the dysregulation of Cdk5 may contribute to disease pathogenesis by perturbing cellular Ca(2+) signaling.

Expression of p25, an aberrant cyclin-dependent kinase 5 activator, stimulates basal secretion in PC12 cells.

Although alterations in the functions of neurotransmitter systems have been implicated in the pathology of Alzheimer’s disease (AD), the mechanisms that give rise to these alterations are not well understood. The amount of p25, an aberrant cleavage product of p35 that activates cyclin-dependent kinase 5 (Cdk5), is elevated in AD brains. The role of Cdk5 in neurotransmitter release has been well established. In this study, we examined whether p25 was linked to altered neurotransmitter release in AD. Transient or stable expression of p25 significantly increased basal secretion of human growth hormone (hGH) or neurotransmitter in PC12 cells. Expression of a p25 phosphorylation-deficient mutant, T138A, inhibited basal hGH secretion relative to the p25 wild type, suggesting the involvement of Thr138 phosphorylation in secretion. The expression and activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), a key protease in the generation of β-amyloid, are increased in AD brains. Our previous studies indicated that overexpression of BACE1 enhanced basal secretion of hGH in PC12 cells. Transient coexpression of p25 and BACE1 further stimulated spontaneous basal secretion. These results indicate a novel role for p25 in the secretory pathway and suggest that elevated levels of p25 and BACE1 in AD brains may contribute to altered neurotransmitter pathology of AD through enhancing spontaneous basal secretion.