Seonghan Kim

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.

α-Synuclein regulates neuronal survival via Bcl-2 family expression and PI3/Akt kinase pathway

α‐Synuclein (α‐SN) is a ubiquitous protein that is especially abundant in the brain and has been postulated to play a central role in the pathogenesis of Parkinsons disease, Alzheimers disease, and other neurodegenerative disorders. However, little is known about the neuronal functions of α‐SN and the molecular and cellular mechanisms underlying neuronal loss. Here, we show that α‐SN plays dual roles of neuroprotection and neurotoxicity depending on its concentration or level of expression. At nanomolar concentrations, α‐SN protected neurons against serum deprivation, oxidative stress, and excitotoxicity through the PI3/Akt signaling pathway, and its protective effect was increased by Bcl‐2 overexpression. Conversely, at both low micromolar and overexpressed levels in the cell, α‐SN resulted in cytotoxicity. This might be related to decreased Bcl‐xL expression and increased bax expression, which is subsequently followed by cytochrome c release and caspase activation and also by microglia‐mediated inflammatory responses via the NFκB and mitogen‐activated protein kinase pathways.

Phosphorylation of amyloid precursor protein (APP) at Thr668 regulates the nuclear translocation of the APP intracellular domain and induces neurodegeneration.

ABSTRACT Amyloid precursor protein (APP) has eight potential phosphorylation sites in its cytoplasmic domain. Recently, it has demonstrated that the constitutive phosphorylation of APP at T668 (APP695 isoform numbering) was observed specifically in the brain. Neuron-specific phosphorylation of APP at T668 is thought to be important for neuronal functions of APP, although its exact physiological significance remains to be clarified. In this study, we show that the phosphorylation of the APP intracellular domain (AICD) at T668 is essential for its binding to Fe65 and its nuclear translocation and affects the resultant neurotoxicity, possibly mediated through the induction of glycogen synthase kinase 3β and tau phosphorylation by enhancing the formation of a ternary complex with Fe65 and CP2 transcription factor. Taken together, these results suggest that the phosphorylation of AICD at T668 contributes to the neuronal degeneration in Alzheimers disease (AD) by regulating its translocation into the nucleus and then affects neurodegeneration; therefore, the specific inhibitor of T668 phosphorylation might be the target of AD therapy.

Effects of the monomeric, oligomeric, and fibrillar Aβ42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone

The incidence of amyloid plaques, composed mainly of β‐amyloid peptides (Aβ), does not correlate well with the severity of neurodegeneration in patients with Alzheimer’s disease (AD). The effects of Aβ42 on neurons or neural stem cells (NSCs) in terms of the aggregated form remain controversial. We prepared three forms of oligomeric, fibrillar, and monomeric Aβ42 peptides and investigated their effects on the proliferation and neural differentiation of adult NSCs, according to the degree of aggregation or concentration. A low micromolar concentration (1 μmol/L) of oligomeric Aβ42 increased the proliferation of adult NSCs remarkably in a neurosphere assay. It also enhanced the neuronal differentiation of adult NSCs and their ability to migrate. These results provide us with valuable information regarding the effects of Aβ42 on NSCs in the brains of patients with AD.

α-Synuclein induces apoptosis by altered expression in human peripheral lymphocyte in Parkinson’s disease

Though the etiology of Parkinson’s disease (PD) remains unclear, α‐synuclein (α‐SN) is regarded as a major causative agent of PD. Several lines of evidence indicate that immunological abnormalities are associated with PD for unknown reasons. The present study was performed to assess whether peripheral blood mononuclear cells (PBMCs) show altered α‐SN expression in PD patients and to identify its functions, which may be related to peripheral immune abnormalities in PD. α‐SN was found to be expressed more in 151 idiopathic PD (IPD) patients than in 101 healthy controls, who nevertheless showed as age‐dependent increases. By in vitro transfection, α‐SN expression was shown to be correlated with glucocorticoid sensitive apoptosis, possibly caused by the enhanced expression of glucocorticoid receptor (GR), caspase activations (caspase‐8, caspase‐9), CD95 up‐regulation, and reactive oxygen species (ROS) production. An understanding of the correlation between α‐SN levels and apoptosis in the presence of the coordinated involvement of multiple processes would provide an insight into the molecular basis of the disease. The present study provides a clue that the α‐SN may be one of the primary causes of the immune abnormalities observed in PD and offers new targets for pharmacotherapeutic intervention.

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.

Mefenamic acid shows neuroprotective effects and improves cognitive impairment in in vitro and in vivo Alzheimer's disease models.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert anti-inflammatory, analgesic, and antipyretic activities and suppress prostaglandin synthesis by inhibiting cyclooxygenase, an enzyme that catalyzes the formation of prostaglandin precursors from arachidonic acid. Epidemiological observations indicate that the long-term treatment of patients suffering from rheumatoid arthritis with NSAIDs results in reduced risk and delayed onset of Alzheimers disease. In this study, we investigated the therapeutic potential for Alzheimers disease of mefenamic acid, a commonly used NSAID that is a cyclooxygenase-1 and 2 inhibitor with only moderate anti-inflammatory properties. We found that mefenamic acid attenuates the neurotoxicities induced by amyloid β peptide (Aβ)1–42 treatment and the expression of a Swedish double mutation (KM595/596NL) of amyloid precursor protein (Swe-APP) or the C-terminal fragments of APP (APP-CTs) in neuronal cells. We also show that mefenamic acid decreases the production of the free radical nitric oxide and reduces cytochrome c release from mitochondria induced by Aβ1–42, Swe-APP, or APP-CTs in neuronal cells. In addition, mefenamic acid up-regulates expression of the antiapoptotic protein Bcl-XL. Moreover, our study demonstrates for the first time that mefenamic acid improves learning and memory impairment in an Aβ1–42-infused Alzheimers disease rat model. Taking these in vitro and in vivo results together, our study suggests that mefenamic acid could be used as a therapeutic agent in Alzheimers disease.

BT-11 improves stress-induced memory impairments through increment of glucose utilization and total neural cell adhesion molecule levels in rat brains

In Oriental medicine, roots of Polygala tenuifolia Willdenow have been known to be an important herb that exhibits sedative effects in insomnia, palpitation with anxiety, restlessness, and disorientation in humans. We previously reported that BT‐11, extracted from those roots, improved scopolamine‐induced amnesia in rats and inhibited acetylcholinesterase activities in vitro. Therefore, we proposed that BT‐11 could remedy stress‐induced memory deficits in rats. In this study, the stress‐induced memory impairments in rats were significantly reversed almost to the control level by BT‐11 treatment. To seek an active component of BT‐11 that plays an important role in antipsychotic effects, we compared BT‐11 with 3,4,5‐trimethoxycinnamic acid (TMCA), which is a constituent of those root extracts. However, the effects of TMCA were less or were not consistent with those of BT‐11 in some of tests. In perticular, BT‐11 reversed the stress‐induced reduction of glucose utilization by [18fluorodeoxyglucose]FDG‐PET and the levels of neural cell adhesion molecule (NCAM) in rat brains to the control levels, whereas TMCA did not. Therefore, BT‐11 improved stress‐induced memory impairments through increment of glucose utilization and total NCAM levels in rat brains. In conclusion, BT‐11 may be strongly effective against stress‐induced amnesia in rats, through the combined effects of TMCA and other active components of BT‐11.

α-Synuclein induces migration of BV―2 microglial cells by up-regulation of CD44 and MT1―MMP

Although there is known to be a marked concentration of reactive microglia in the substantia nigra pars compacta (SNpc) of patients with Parkinson’s disease (PD), a disorder in which α‐synuclein plays a key pathogenic role, the specific roles of α‐synuclein and microglia remains poorly understood. In this study, we investigated the effects of α‐synuclein and the mechanisms of invasive microglial migration into the SNpc. We show that α‐synuclein up‐regulates the expressions of the cell adhesion molecule CD44 and the cell surface protease membrane‐type 1 matrix metalloproteinase through the extracellular regulated kinases 1/2 pathway. These concurrent inductions increased the generation of soluble CD44 to liberate microglia from the surrounding extracellular matrix for migration. The effects of α‐synuclein were identical in BV‐2 murine microglial cells subjected to cDNA transfection and extracellular treatment. These inductions in primary microglial cultures of C57Bl/6 mice were identical to those in BV‐2 cells. α‐Synuclein‐induced microglial migration into the SNpc was confirmed in vivo using a 6‐hydroxydopamine mouse model of PD. Our data demonstrate a correlation between α‐synuclein‐induced phenotypic changes and microglial migration. With the recruitment of the microglial population into the SNpc during dopaminergic neurodegeneration, α‐synuclein may play a role in accelerating the pathogenesis of PD.

Role of follicular dendritic cells in the apoptosis of germinal center B cells

Follicular dendritic cells (FDCs) provide the most obvious source of antigens, which are essential for the differentiation of GC B cells. It has been reported that most proliferating B cells in germinal centers undergo apoptosis. Quantitative histology shows macrophages with apoptotic debris throughout the germinal center, the highest frequency of these cells being found in the dense FDC network. Based on these findings, we hypothesized that FDC may be involved in an apoptotic pathway of the germinal center B cells. To prove this hypothesis, we performed double immunohistochemical analysis using anti-FDC mAb and peanut agglutinin (PNA), with their respective TUNEL kits. Collated data showed that a great proportion of the apoptotic cells, most of which were positive for PNA, were in close contact with FDC, which indicated an interaction between FDC and B cells in the apoptotic pathway. Further studies using double immunohistochemical staining and FACS analyses demonstrated the expression of Fas-ligand (FasL) in a subset of the FDC. These results suggest that FDC may play a role in the apoptosis of germinal center B cells via Fas-FasL interaction.