Young Hae Chong

C-terminal fragments of amyloid precursor protein exert neurotoxicity by inducing glycogen synthase kinase-3β expression

The AICD (amyloid precursor protein [APP] intracellular domain) and C31, the caspase‐cleaved C‐terminal fragment of APP, have been found in the brains of patients with Alzheimers disease (AD). Here, we demonstrate for the first time that the C‐terminal fragments of APP (AICD [C57, C59] and C31) exert neurotoxicity on differentiated PC 12 cells and rat primary cortical neurons by inducing the expression of glycogen synthase kinase 3β, forming a ternary complex with Fe65 and CP2/LSF/LBP1 in the nucleus, whereas deletion mutants and a point mutant with Y682G of the YENPTY domain, a Fe65 binding domain, do not. Moreover, expression of APP770 and Swedish mutant form of APP increased the levels of C‐terminal fragments of APP (APP‐CTs) in neuronal cells and also induced the up‐regulation of glycogen synthase kinase‐3β at both the mRNA and the protein levels. In addition, we show that CP2/LSF/LBP1 binding site (nt +0 to ∼+10) in human glycogen synthase kinase 3β promoter region is essential for the induction of the gene transcription by APP‐CTs. The neurotoxicities induced by APP‐CTs (AICD and C31) were accompanied by an increase in the active form of glycogen synthase knase‐3β, and by the induction of tau phosphorylation and a reduction in nuclear β‐catenin levels, and led to apoptosis.

ERK1/2 activation mediates Aβ oligomer-induced neurotoxicity via caspase-3 activation and Tau cleavage in rat organotypic hippocampal slice cultures

In this study, we investigated the molecular basis for the altered signal transduction associated with soluble amyloid β-protein (Aβ) oligomer-mediated neurotoxicity in the hippocampus, which is primarily linked to cognitive dysfunction in Alzheimer disease (AD). As measured by media lactate dehydrogenase levels, and staining with propidium iodide, acute exposure to low micromolar concentrations of the Aβ1-42 oligomer significantly induced cell death. This was accompanied by activation of the ERK1/2 signal transduction pathway in rat organotypic hippocampal slices. Notably, this resulted in caspase-3 activation by a process that led to proteolytic cleavage of Tau, which was recently confirmed to occur in AD brains. Tau cleavage likely occurred in the absence of overt synaptic loss, as suggested by the preserved levels of synaptophysin, a presynaptic marker. Moreover, among the pharmacological agents tested to inhibit several kinase cascades, only the ERK inhibitor significantly attenuated Aβ1-42 oligomer-induced toxicity concomitant with the reduction of activation of ERK1/2 and caspase-3 to a lesser extent. Importantly, the caspase-3 inhibitor also decreased Aβ oligomer-induced cell death, with no appreciable effect on the ERK signaling pathway, although such treatment was effective in reducing caspase-3 activation and Tau cleavage. Therefore, these results suggest that local targeting of the ERK1/2 signaling pathway to reduce Tau cleavage, as occurs with the inhibition of caspase-3 activation, may modulate the neurotoxic effects of soluble Aβ oligomer in the hippocampus and provide the rationale for symptomatic treatment of AD.

Mechanisms involved in prostaglandin E2-mediated neuroprotection against TNF-α: possible involvement of multiple signal transduction and β-catenin/T-cell factor

Abstract Cerebrospinal fluid prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-α) levels are elevated in patients with Alzheimers disease (AD), which suggests that they are involved in neurodegeneration. We previously reported that TNF-α derived from human macrophages, in response to β-amyloid or amyloidogenic C-terminal peptide, is a main mediator of inflammatory neurotoxicity. In a continuation of this work, the present study investigated the direct effect of PGE2, one of the major prostaglandins produced in the brain, on cell viability in SH-SY5Y neuronal cells treated with TNF-α. PGE2 did not promote neurotoxicity, but rather had a strong protective effect against TNF-α by ameliorating TNF-α-induced apoptosis and also by rescuing the intracellular level of β-catenin, a key transducer of the Wnt signaling pathway. PGE2-mediated stabilization of β-catenin was accompanied by T-cell factor/lymphoid enhancer factor (Tcf/Lef)-mediated transcriptional activation, which was followed by an increase in the cyclinD1 level. Pharmacological studies provided further evidence supporting the notion that PGE2-mediated neuroprotection against TNF-α involves the stimulation of Tcf/Lef signaling through EP1-, EP2-, and EP4-mediated increases of β-catenin in SH-SY5Y cells. In addition, this PGE2 effect appears to be dependent on the activation of protein kinase A, phosphatidylinositol 3-kinase, phospholipase C, and to a lesser extent protein kinase C. Thus, the molecular mechanism governing the inhibitory effect of PGE2 against TNF-α may involve the activation and cross talk of multiple signal transduction and play an important role in regulating the survival of neurons during the neurotoxic inflammatory response associated with neurodegenerative diseases including AD.

S100a9 Knockdown Decreases the Memory Impairment and the Neuropathology in Tg2576 Mice, AD Animal Model

Inflammation, insoluble protein deposition and neuronal cell loss are important features in the Alzheimers disease (AD) brain. To investigate the regulatory genes responsible for the neuropathology in AD, we performed microarray analysis with APPV717I-CT100 transgenic mice, an animal model of AD, and isolated the S100a9 gene, which encodes an inflammation-associated calcium binding protein. In another AD animal model, Tg2576 mouse brain, and in human AD brain, induction of S100a9 was confirmed. The endogenous expression of S100a9 was induced by treatment with Aβ or CT peptides in a microglia cell line, BV2 cells. In these cells, silencing study of S100a9 showed that the induction of S100a9 increased the intracellular calcium level and up-regulated the inflammatory cytokines (IL-1β and TNFα) and iNOS. S100a9 lentiviral short hairpin RNA (sh-S100a9) was injected into the hippocampus region of the brains of 13-month-old Tg2576 mice. At two months after injection, we found that knockdown of S100a9 expression had improved the cognition decline of Tg2576 mice in the water maze task, and had reduced amyloid plaque burden. These results suggest that S100a9 induced by Aβ or CT contributes to cause inflammation, which then affects the neuropathology including amyloid plaques burden and impairs cognitive function. Thus, the inhibition of S100a9 is a possible target for AD therapy.

Inhibiting Mer receptor tyrosine kinase suppresses STAT1, SOCS1/3, and NF-κB activation and enhances inflammatory responses in lipopolysaccharide-induced acute lung injury

Mer signaling participates in a novel inhibitory pathway in TLR activation. The purpose of the present study was to examine the role of Mer signaling in the down‐regulation of TLR4 activation‐driven immune responses in mice, i.t.‐treated with LPS, using the specific Mer‐blocking antibody. At 4 h and 24 h after LPS treatment, expression of Mer protein in alveolar macrophages and lung tissue decreased, sMer in BALF increased significantly, and Mer activation increased. Pretreatment with anti‐Mer antibody did not influence the protein levels of Mer and sMer levels. Anti‐Mer antibody significantly reduced LPS‐induced Mer activation, phosphorylation of Akt and FAK, STAT1 activation, and expression of SOCS1 and ‐3. Anti‐Mer antibody enhanced LPS‐induced inflammatory responses, including activation of the NF‐κB pathway; the production of TNF‐α, IL‐1β, and MIP‐2 and MMP‐9 activity; and accumulation of inflammatory cells and the total protein levels in BALF. These results indicate that Mer plays as an intrinsic feedback inhibitor of the TLR4‐ and inflammatory mediator‐driven immune responses during acute lung injury.

N-acetylcysteine inhibits RhoA and promotes apoptotic cell clearance during intense lung inflammation.

RATIONALE The resolution of pulmonary inflammation seen in various inflammatory lung conditions depends on the clearance of apoptotic cells to prevent permanent tissue damage or progressive disease. Uptake of apoptotic cells by alveolar macrophages is suppressed by oxidants through the activation of Rho signaling. OBJECTIVES We hypothesized that antioxidant exposure would increase the ability of alveolar macrophages to clear pulmonary apoptotic cells through the inhibition of RhoA. METHODS The effects of the antioxidant N-acetylcysteine (NAC) on the pulmonary immune response were seen in mice treated intratracheally with LPS, LPS + NAC, or saline. Apoptotic cell clearance, RhoA activity, and changes in the lung inflammatory responses were analyzed in vivo or ex vivo. MEASUREMENTS AND MAIN RESULTS Neutrophil accumulation, apoptosis, necrosis, and oxidant production peaked at 3 days post LPS treatment. NAC enhanced the clearance of apoptotic cells and inhibited RhoA activity in alveolar macrophages at 3 days post LPS treatment. NAC suppressed LPS-induced proinflammatory mediators, enhanced the production of transforming growth factor-beta1, reduced the accumulation of inflammatory cells, and reduced levels of protein and lactate dehydrogenase in bronchoalveolar lavage fluid. In the presence of ex vivo apoptotic cells, alveolar macrophages exposed to LPS or LPS + NAC had reduced tumor necrosis factor-alpha levels and increased transforming growth factor-beta1 levels. A Rho kinase inhibitor mimicked the effects of NAC on the clearance of apoptotic cells and the inflammatory responses. CONCLUSIONS These results indicate that NAC can expedite the resolution of LPS-induced pulmonary inflammation through the inhibition of RhoA activity and the enhancement of apoptotic cell clearance.

Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

BackgroundAlthough in vitro studies have determined that the activation of mitogen-activated protein (MAP) kinases is crucial to the activation of transcription factors and regulation of the production of proinflammatory mediators, the roles of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in acute lung injury have not been elucidated.MethodsSaline or lipopolysaccharide (LPS, 6 mg/kg of body weight) was administered intratracheally with a 1-hour pretreatment with SP600125 (a JNK inhibitor; 30 mg/kg, IO), or PD98059 (an MEK/ERK inhibitor; 30 mg/kg, IO). Rats were sacrificed 4 hours after LPS treatment.ResultsSP600125 or PD98059 inhibited LPS-induced phosphorylation of JNK and ERK, total protein and LDH activity in BAL fluid, and neutrophil influx into the lungs. In addition, these MAP kinase inhibitors substantially reduced LPS-induced production of inflammatory mediators, such as CINC, MMP-9, and nitric oxide. Inhibition of JNK correlated with suppression of NF-κB activation through downregulation of phosphorylation and degradation of IκB-α, while ERK inhibition only slightly influenced the NF-κB pathway.ConclusionJNK and ERK play pivotal roles in LPS-induced acute lung injury. Therefore, inhibition of JNK or ERK activity has potential as an effective therapeutic strategy in interventions of inflammatory cascade-associated lung injury.

Norepinephrine differentially modulates the innate inflammatory response provoked by amyloid-β peptide via action at β-adrenoceptors and activation of cAMP/PKA pathway in human THP-1 macrophages.

Evidence indicates that norepinephrine (NE) has antiinflammatory activities and plays a neuroprotective role where inflammatory events contribute to Alzheimers disease pathology. Here, we evaluated the effects of NE on amyloid beta 1-42 (Aβ1-42)-induced cytotoxicity and proinflammatory cytokine/chemokine secretion, and determined the mechanisms through which NE exerts its actions in human THP-1 macrophages. NE clearly reduced the Aβ1-42-mediated production of the proinflammatory chemokine, monocytic chemotactic protein-1 (MCP-1/CCL2). In contrast to its ability to reduce MCP-1 secretion, NE enhanced the amounts of the proinflammatory cytokine interleukin (IL)-1β secreted from Aβ1-42 treated cells. NE significantly reduced the Aβ1-42-induced cytotoxicity in situations where it contributed to the increased IL-1β and decreased MCP-1 during Aβ1-42 stimulation. The ability of NE to differentially modulate the Aβ1-42-induced immune responses was mediated by β-adrenoceptors, as the aforementioned effects were replicated by the β-adrenoceptor agonist, isoproterenol, and blocked by the β-adrenoceptor antagonist, dl-propranolol. Of note, the NE effects on Aβ1-42-induced responses were mimicked by dbcAMP and forskolin, but significantly blocked by H89, an inhibitor of PKA. Moreover, NE abolished Aβ1-42-mediated decline of CREB phosphorylation. Overall, NE suppresses Aβ1-42-mediated cytotoxicity and MCP-1 secretion, but enhances Aβ-mediated IL-1β secretion through action at β-adrenoceptors, accompanied by activation of cAMP/PKA pathway and CREB in human microglia-like THP-1 cells.

Resveratrol reduces glutamate-mediated monocyte chemotactic protein-1 expression via inhibition of extracellular signal-regulated kinase 1/2 pathway in rat hippocampal slice cultures

J. Neurochem. (2010) 112, 1477–1488.

Amyloidogenic processing of Alzheimer's amyloid precursor protein in vitro and its modulation by metal ions and tacrine

Recent studies implicate that excessive amyloidogenic pathway of amyloid precursor protein (APP) processing may be the final common pathway involved in the pathogensis of AD. In attempts to identify the proteases or factors leading to excessive amyloid deposition, we evaluated the potential role of acethylcholinesterase (AChE) and its associated protease for amyloidogenic processing of APP in vitro. Prolonged incubation of a recombinant APP770 with AChE produced several amyloidogenic fragments accumulating a relatively stable a 18 kDa A beta (amyloid beta-protein) bearing carboxy terminal peptide, which was further degraded by an increased concentration of AChE. Protease inhibitory profiles confirmed the trypsin-like serine protease activity present in AChE preparation. This observed APP processing was significantly enhanced by Ca2+, Mg2+, or Mn2+ at 1 mM concentration and modulated in concentration dependent manners by metal ions such as Ca2+, Zn2+, Fe2+/Fe3+, Al3+, or a tacrine, a centrally active cholinesterase inhibitor. Our data imply that AChE and its associated protease may be involved in the generation a 18 kDa amyloidogenic peptide under certain physiological condition in vivo and that the gradual changes in their proteolytic activities or locations and the locally disturbed metal homeostasis could be factors associated with abnormal accumulation of APP, eventually leading to amyloid deposition in AD brain. In addition, zinc or tacrine treatment of AD patients with high dosage or in the long term may have effects on the process of amyloidogensis.