Ji-Heui Seo

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.

Novel cognitive improving and neuroprotective activities of Polygala tenuifolia Willdenow extract, BT-11.

We carried out this study to search a new active constituent that had cognitive enhancing activity and low side effects from natural source. We found that the extract of dried root of Polygala tenuifolia Willdenow (BT‐11, 10 mg/kg, i.p.) could significantly reverse scopolamine‐induced cognitive impairments in rat, using a passive avoidance and a water maze test. We also investigated the effects of BT‐11 on neurotoxicity induced by glutamate (Glu) and toxic metabolites of amyloid precursor protein (APP) such as amyloid β protein (Aβ) and C‐terminal fragment of APP (CT) in primary cultured neurons of rat. The pretreatment of BT‐11 (0.5, 3, and 5 μg/ml) significantly reduced cell death induced by Glu (1 mM), Aβ (10 μM) and CT105 (10 μM) in a dose‐dependent manner. In addition, BT‐11 inhibited acetylcholinesterase (AChE) activity in a dose‐dependent and non‐competitive manner (IC50 value; 263.7 μg/ml). Our novel findings suggest the possibility that this extract may have some protective effects against neuronal death and cognitive impairments in Alzheimers disease (AD), or other neurodegenerative diseases related to excitotoxicity and central cholinergic dysfunction.

Amyloid β peptide induces cytochrome c release from isolated mitochondria

Amyloid &bgr; peptide (A&bgr;) is a neurotoxic metabolic product of the amyloid precursor protein (APP). A&bgr; is strongly implicated in the pathology of Alzheimers disease (AD) and can be formed intracellularly. In this study, we show that the addition of A&bgr;1-42 to isolated mouse brain mitochondria can directly induce cytochrome c (Cyt c) release and mitochondrial swelling, which were partially inhibited by cyclosporin A (CsA). These results suggest that the A&bgr;accumulated intracellularly by APP processing might exert neurotoxicity by interacting with mitochondria and inducing mitochondrial swelling and release of Cyt c, which activates caspase-3 and finally can lead to apoptosis in neuronal cells and to neurodegeneration in 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.

Memory impairment and cholinergic dysfunction by centrally administered Aβ and carboxyl-terminal fragment of Alzheimer’s APP in mice

Alzheimers disease (AD) is characterized by cognitive impairment and excessive deposition of neurite plaques (NPs). Although the pathogenic mechanism of AD is not exactly known, there is increasing evidence that amyloid beta protein (Aβ), which is the major constituent of NPs and is derived from amyloid precursor protein (APP), plays a key role in the neuronal degeneration in AD. However, because there are several discrepancies between Aβ deposition and neurodegeneration or cognitive impairment in AD, the possible effects of other cleavage products of APP need to be explored. We previously showed that a recombinant carboxyl‐terminal 105 amino acid fragment of APP (CT105) induced neurotoxicities both in vitro and in vivo. However, it is unclear whether learning and memory impairments and neurochemical changes are induced by CT 105. To elucidate the in vivo neurotoxicities of Aβ and CT105, we examined behavioral changes using a Y‐maze and a water maze task following a single intracerebroventricular injection of CT105 (68.5, 342 and 685 pmol) or Aβ1–42 (685 pmol) to mice. We also quantified the changes in the acetylcholine (ACh) level and assayed mitochondrial pyruvate dehydrogenase (PDH) activity. CT105 caused much more significant impairments in cued, spatial, and working memory performances of mice in Y‐maze and water maze tasks in a dose‐dependent manner than Aβ1–42. Also, the mice injected with CT105 revealed significant decreases in ACh levels and reduced PDH activities in the cerebral cortex and hippocampus compared with the saline‐treated control and Aβ1–42‐treated groups. These results suggest that learning and memory impairment induced by centrally administered CT105 is in some way related to the reduced ACh levels and mitochondrial PDH activities and that CT105 is more potent than Aβ in inducing cognitive dysfunction.

Effects of nicotine on APP secretion and Aβ- or CT105-induced toxicity

Several lines of evidence indicated that overexpression or aberrant processing of amyloid precursor protein (APP) is causally related to Alzheimers disease (AD). Amyloid precursor protein is principally cleaved within the amyloid beta protein domain to release a large soluble ectodomain (APPs), known to have a wide range of trophic functions. The central hypothesis guiding this review is that nicotine may play an important role in APP secretion and protection against toxicity induced by APP metabolic fragments (beta-amyloid [Abeta], carboxyl terminal [CT]). Findings from our experiments have shown that nicotine enhances the release of APPs, which has neurotrophic and neuroprotective activities in concentration-dependent (>50 micromol/L) and time-dependent (>2 hours) manners. In addition, pretreatment of nicotine (>10 micromol/L for 24 hours) partially prevented Abeta or CT(105)-induced cytotoxicity in primary cultured neuron cells, and the effects of nicotine-induced protection were inhibited by the pretreatment with a nicotine alpha-bungarotoxin. Nicotine (>10 micromol/L for 24 hours) partially inhibited CT(105)-induced cytotoxicity when PC12 cells was transfected with CT(105). From these results, we proposed that nicotine or nicotinic receptor agonist treatment might improve the cognitive functions not only by supplementation of cholinergic neurotransmission, but also by protecting Abeta- or CT(105)-induced neurotoxicity probably through the increased release of APPs and the activation of nicotinic receptors.

Glutamate and aspartate impair memory retention and damage hypothalamic neurons in adult mice

We examined the effects of systemic administration of monosodium glutamate (MSG) or aspartate (ASP) on the memory retention and neuronal damage in the brains of adult mice. Compared with the control mice, a single intraperitoneal injection of either 4.0 mg/g MSG or 0.5 mg/g ASP after acquisition trial significantly shortened the response latency in the passive avoidance test, accompanying by the transient weight loss. Histopathological analysis of the brains of these mice revealed that neurons in the arcuate nucleus of hypothalamus were damaged markedly by MSG (4.0 mg/g) or ASP (0.5 mg/g). Other brain areas including cerebral cortex and hippocampus did not show any pathological changes. These findings suggest that systemic administration of MSG or ASP could impair memory retention and damage hypothalamic neurons in adult mice.

A Specific Need for CRKL in p210BCR-ABL–Induced Transformation of Mouse Hematopoietic Progenitors

CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL), the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear, however, whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here, we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore, a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation.

Effects of carboxyl-terminal fragment of Alzheimer’s amyloid precursor protein and amyloid β-peptide on the production of cytokines and nitric oxide in glial cells

In a brain with Alzheimers disease (AD), activated glial cells are observed surrounding amyloid plaque. In this study, to investigate the roles of carboxyl‐terminal fragments (CTs) of amyloid precursor protein (APP) and amyloid β‐peptide (Aβ) in inflammatory processes possibly linked to neurodegeneration associated with AD, we examined the effects of the CT of APP with 105 amino acid residues (CT105) on the alteration of inflammatory mediators in rat cortical astrocytes and microglial cells and compared these effects to those of Aβ. We found that cytokines including interleukin 1β (IL‐1β) and tumor necrosis factor a and chemokines such as macrophage inflammatory protein 1a, monocyte chemoattractant protein 1, and RANTES (regulated on activation, normal T expressed and secreted) were highly induced by 100 nM CT105 in a time‐dependent manner, whereas the same amount of Aβ1‐42 did not significantly induce these mediators. In addition, inducible nitric oxide synthase (iNOS) expression and nitrite accumulation induced by CT105 were inhibited by IL‐1 receptor antagonist (IL‐1ra). Furthermore, the addition of conditioned media from CT105‐treated astrocytes to rat cortical neurons caused a decrease in cell viability. Pretreatment with IL‐1ra, NG‐nitro‐L‐arginine methyl ester (a specific inhibitor of NOS), and pyrollidine dithiocarbamate [an inhibitor of nuclear factor‐κB (NF‐κB)] prevented neuronal cell death caused by conditioned media from CT105‐ treated astrocytes. Our results indicate that the activation of astrocytes by CT105 leads to neuronal cell death in vitro, presumably caused by the increase in nitric oxide derived from the induction of iNOS through the activation of cytokines and NF‐κB. Thus, CT105 may contribute to stimulation of inflammatory processes linked to delayed neurodegeneration in AD.