Neurodegenerative diseases: Alzheimer's disease

Neurodegenerative diseases: Alzheimer’s diseaseP21-1Altered trace elemental homeostasis in neuro-degenerative (Alzheimer’s and Parkinson’s) and neuro-psychiatric (bipolar) disorderP. Shanmugavelu,* T. S. Sathyanarayana Rao,� M. L. Hegde,�M.S. Mustak,� R. B. Menon,* R. V. Rao* and K. S. Jagannatha

Rao�

*Analytical Control Section, Bhabha Atomic Research Centre,

Mumbai; �Department of Psychiatry, J.S.S. Medical College andHospital, Mysore; �Department of Biochemistry and Nutrition,Central Food Technological Research Institute, Mysore, India

Data on the level of trace elements in different vital organs in control and

pathological conditions is very essential to establish a cause and effect relationship

in brain. Trace level elements were analysed by ICPAES in brain and CSF of

Alzheimer’s disease (AD), serum samples of Parkinson’s disease (PD) and bipolar

disorder (BD). The results showed imbalance in trace elemental homeostasis. In

moderately affected AD brain, monovalent elements were significantly reduced,

while divalent elements were increased. In severely affected AD brains, Al and Fe,

and S were predominantly present displacing monovalent and divalent elements. It

was found that Zn accumulates more in moderate, while Al and Fe deposition were

more in severely affected AD. In case of AD, CSF samples, levels of K, P, and S

were decreased significantly, while Na was increased compared with control. In

both early and severe PD serum, Al and S were decreased compared with control.

Fe and Zn were significantly low in severe PD, while K, Mg, Cu, Co and P were

high in early and severe PD compared with control. In BD groups (I, II and V) Na,

P, Cu, Al and Mn were elevated significantly, while S, Fe, and Zn were decreased

significantly. The observations are novel and significant.

Keywords: Alzheimer’s disease, bipoar disorders, neurodegeneration, trace

metals.

P21-2Loss of serotonin 5-HT2A receptors in the temporalcortex correlates with cognitive decline in Alzheimer’sdiseaseK. P. Lai, S. W. Tsang, J. Keene, T. Hope, M. M. Esiri, P. T. Francis

and C. P. Chen

Department of Clinical Research, Singapore General Hospital,

Singapore; Department of Pharmacology, National University of

Singapore, Singapore; Department of Psychiatry, Warneford

Hospital, Oxford, UK; Department of Neuropathology, Radcliffe

Infirmary, Oxford, UK; Wolfson Centre for Age-Related Diseases,

King’s College, London, UK; National Neuroscience Institute,

Singapore

Background: Postmortem and imaging studies have demonstrated reductions of

serotonin 5-HT2A receptors in the neocortex of Alzheimer’s disease (AD) patients.

However, it is unclear whether such losses play a role in the clinical features of

AD.

Objective: To correlate 5-HT2A receptor status with clinical features in AD.

Methods: A total of 30 patients with AD were prospectively assessed for cognitive

and behavioral changes from study entry till death. 5-HT2A receptors in the

postmortem orbitofrantal and mid temporal cortices were measured by saturation

radioligand binding assays.

Results: 5-HT2A receptor densities were reduced in AD compared with aged

controls. In the AD group, more severe loss of 5-HT2A receptors in the mid-

temporal cortex correlated with higher rates of decline of both mini-mental state

examination (MMSE) and Rivermead Behavioural Memory Test (RBMT) scores.

These associations were found to be independent of the effects of choline

acetyltransferase (ChAT) activity and presence of behavioral symptoms.

Conclusions: We propose that patients with AD who have greater losses of 5-

HT2A receptors may be predisposed to faster cognitive decline. These findings

suggest that serotomimetics may be useful adjuvants to cholinergic replacement

therapies.

Keywords: learning and memory, neurodegenerative diseases – Alzheimer’s,

neurotransmitters, receptors.

P21-3Multiplicity effects of oxidative stress in the cortex of atransgenic mouse model of Alzheimer’s diseaseJ. K. Yao, P. Cheng, T. M. Wengenack and J. F. Poduslo

VA Pittsburgh Healthcare System and University of Pittsburgh

Medical Center, Pittsburgh, PA; Mayo Clinic, Rochester, MN, USA

Accumulating evidence supports an etiologic heterogeneity for Alzheimer’s

disease (AD), in which case there probably exists a final common pathogenetic

pathway. To determine the multiplicity consequences of redox imbalance, we have

developed a high-pressure liquid chromatography procedure coupled with a

Coulometric Multi-Electrode Array System to evaluate a network of multiple

interacted biochemical pathways. The aim of this study is to assess whether

multiplicity effects of oxidative stress present in the double transgenic mice

expressing both mutant human b-amyloid precursor protein (APP) and presenilin 1

(PS1). Specifically, we compare glutathione (GSH) redox state, purine catabolism,

monoamine metabolites, antioxidants and oxidative damage products simulta-

neously in the cortex of APP, PS1 tansgenics compared with wild type (WT) B6/

SJL mice at ages 4- and 9-months. The ratio of GSSG (oxidized GSH) to GSH was

significantly higher pb peptide (Ab)-induced oxidative stress in the AD pathology.

Moreover, there exist multiplicity consequences of redox imbalance that mediates

the recognizable syndromes of AD. Ab-induced oxidative stress may be detected

in the early course of AD.

Keywords: Alzheimer’s disease, cortex, glutathione, oxidative stress, transgenic

mouse.

P21-4Phosphorylation-dependent regulation of C-terminalfragments of APP in translocation into the nucleusK.-A. Chang, J.-W. Ha, H.-S. Kim and Y.-H. Suh

Department of Pharmacology, NatOl Creative Res. Initiative Cent.

for AD, Coll. of Med, Seoul Nat’l Univ., Seoul, Korea

APP has been known to be phosphorylated at eight serine and threonine residues in

its cytoplasmic domain in neuronal cells. Among them, the phosphorylation of

Thr668 has been reported to result in the significant conformational change that

may affect the interactions of APP with its binding partners. We have previously

reported that AICD and C31 (the caspase-cleaved form of APP) induce glycogen

synthase kinase 3 beta (GSK-3b) expression, forming a ternary complex with Fe65

and CP2/LSF/LBP1 transcription factor in the nucleus of NGF-differentiated PC

12 cells and rat primary cortical neurons. Here we demonstrated that the

phosphorylation of APP-CTs at Thr668 is essential for their binding to Fe65 and

that the inhibition of the phosphorylation of APP at Thr668 by site-directed

mutagenesis (T668A) prevented the translocalization into the nucleus with Fe65,

inhibiting the up-regulation of GSK-3b induced by APP-CTs. We also show that

significantly increased pAPP (Thr668) and GSK-3b are detected in the brains of

AD patients. Taken together, we suggest that APP-CTs might contribute to the

neuronal degeneration in AD through inducing GSK-3b expression by

phosphoryation-dependent manner.

Keywords: cell death and survival, CP2/LSF/LBP1, Fe65, glycogen synthase

kinase 3 beta, neurodegenerative diseases – Alzheimer’s.

� 2004 International Society for Neurochemistry, Journal of Neurochemistry, 88 (Suppl. 1) 49

P21-5Effects of enriched environment training on theprogression of Alzheimer’s disease-like symptoms ofTg2576 transgenic miceP. E. Vivas-Mej’a, J. Felix and S. PeAa de Ortiz

University of Puerto Rico, Rio Piedras

Recently, it has been hypothesized that the level of education and

social stimuli that promote positive behavioral and mental activities

can delay the onset of Alzheimer disease (AD). We used Tg2576

mice (Tg) to test the hypothesis that a long-term enriched

environment (EE) can delay the onset of the pathological features

of AD in these transgenic animals. Wild type (WT, 12 week old)

and Tg animals were used initially in this study. The cycle of EE

stimulation and behavioral tests is repeated six times (until all of the

animals are 12 months old). All groups of animals are subjected to

behavioral tests before and after 3 weeks of stimulation in EE. So

far, the EE does not have noticeable effects neither in WT or Tg

animals, in object recognition memory, a hippocampal independent

task. However, an EE seems to increase the execution of the animals

in place recognition memory and in the holeboard spatial

discrimination task, two hippocampal-dependent tasks. This effect

is especially noticeable in Tg animals. At the end of the six cycles of

EE we will investigate the underlying differences in pathology and

expression of molecular markers between the experimental groups.

Acknowledgements: NIH/NCRR/COBRE programs sponsor this

work.

Keywords: Alzheimer’s, enrich, enviroment, Tg2576.

P21-6Processing APP At The B-site requires proper orientationto be accessed by BACE1R. Yan,*§ I. Qahwash,*� W. He,*§ A. Tomasselli� and

R. F. Kletzien *

*Cell & Molecular Biology; �Protein Sciences, PharmaciaCorporation, Kalamazoo, MI; �Graduate Program, University ofChicago School of Medicine, Chicago, IL; §Department of

Neurosciences, Lerner Research Institute, Cleveland Clinic

Foundation, Cleveland, OH, USA

Membrane-bound BACE1 naturally cleaves its transmembrane

substrate amyloid precursor protein (APP) at the two adjacent beta-

and beta�-site. Cleavages at these two sites bring about the

heterogeneous N-terminal end of APP C-terminal fragments that are

further processed by gamma-secretase to release Abeta1-40/42 or

Abeta11-40/42. The significance underlying Ab11-40/42 in Alzhei-

mer’s pathogenesis has remained to be experimentally elucidated, but

increased productions of Ab1-40/42 have been broadly demonstrated

to contribute amyloid depositions in senile plaques. In this study, we

report that the cleavage of APP at the beta-site by BACE1 is readily

disrupted through limited structural twists while the beta�-site is

relatively better positioned to gain access to the BACE1 catalytic

cavity. Although the loop region of BACE1 confers the mobility of

BACE1 catalytic domain, either lengthening or shortening the loop

region did not impart the selective cleavage of APP at these two

adjacent sites. The knowledge gained from this study will offer an

opportunity for rational design of small molecule drugs to block

specifically the cleavage of APP at the beta-site while not to disturb

the functions of other cellular aspartyl proteases.

Keywords: amyloid peptide, APP processing, BACE1, cleavage

sequence.

P21-7Minocycline blocks C-terminal fragments of APP-inducedneurotoxicity by inhibition cytochrome c release andcaspase-12 activationE.-M. Kim, Y. Choi and Y.-H. Suh

Department of Pharmacology, College of Medicine, National

Creative Research Initiative Center for Alzheimer’s Dementia and

Neuroscience Research Institute, MRC, Seoul National University,

Seoul, South Korea

Minocycline is a second-generation tetracycline that effectively

crosses the blood–brain barrier. It has remarkable neuroprotective

qualities in models of cerebral ischaemia, traumatic brain injury, and

Huntington’s and Parkinson’s disease. However, there is no

evidence about neuroprotective effects of minocycline on AD.

Alzheimer’s disease (AD) is a neurodegenerative disorder char-

acterized neuropathologically by the presence of neuritic plaques

containing amyloid fibrils and neurofibrillary tangles whose main

component is paired helical filament composed of hyperpho-

sphorylated tau. There are numerous lines of evidence that some

of the neurotoxicity associated with AD is because of proteolytic

fragments of the amyloid precursor protein (APP). In this study, we

have obtained that minocycline reduces neurotoxicity induced by

various C-terminal fragments of APP through inhibition of

cytochrome c release and caspase-12 activation.

Keywords: Alzheimer’s disease, C-terminal fragment of amyloid

precursor protein, caspase-12, cytochrome c, minocycline.

P21-8NMDA receptor dysfunction in Alzheimer’s diseaseM. R. Hynd, H. L.Scott and P. R. Dodd

Department of Biochemistry, University of Queensland, St Lucia,

Queensland, Australia

The inherent neurotoxic potential of the endogenous excitatory

amino acid glutamate, may be causally related to the pathogenesis

of AD neurodegeneration disorders. Neuronal excitotoxicity is

conceivably mediated by the N-methyl-D-aspartate-(NMDA)-Ca2+-

ionotropic receptor. NMDA receptors exist as multimeric complexes

comprising proteins from two families – NR1 and NR2(A-D). The

polyamines, spermine and spermidine bind to, and modulate

NMDA receptor efficacy via interaction with exon 5, an

alternatively-spliced, 21 amino acid, N-terminal cassette. AD-

associated cognitive impairment may therefore occur via subunit-

specific NMDA receptor dysfunction effecting regional selectivity

of neuronal degradation. Total RNA was prepared from pathologi-

cally spared and susceptible regions from AD cases and matched

controls. Quantitation was performed using standard curve

methodology in which a known amount of a synthetic ribonucleic

acid competitor deletion construct was co-amplified against total

RNA. Expression profile analysis of two NR1 mRNA subsets has

revealed significant differences in NR11XX mRNA levels in

cingulate gyrus, P.

Keywords: aging, ion channels, neurodegenerative diseases –

Alzheimer’s, neuroprotection, neurotoxicity, receptors.

50 � 2004 International Society for Neurochemistry, Journal of Neurochemistry, 88 (Suppl. 1)

P21-9Streptozotocin-induced Alzheimer’s disease in rats:neurochemical evaluationH. Mehta and K. P. Mohanakumar

Division of Neurosciences, Indian Institute of Chemical Biology,

Calcutta, India.

In sporadic Alzheimer’s disease (AD), a number of metabolic alterations, in

particular, reduced levels of acetylcholine and impairments of glucose

utilization are prominent. Anticholinesterase therapy affects other neuro-

transmitters in addition to the level of acetylcholine in the brain.

Streptozotocin (STZ) was administered intracerebroventricularly (icv) in

the rat brain and we studied the changes in neurotransmitter systems and

behavior, which resembled those seen in AD. Nucleus caudate putamen

(NCP) and hippocampus (HP) were analyzed for acetylcholinesterase

(AChE) and cholineacetyltransferase (ChAT) activity, and for biogenic

amines levels. Cognitive functions were assessed using a Plus-maze

apparatus. AChE activity increased significantly in striatum, whereas reduced

in HP on 21st day following STZ administration. ChAT activity was reduced

significantly in both the regions after 3 weeks of STZ administration.

Dopamine (DA) and serotonin (5-HT) levels were reduced significantly in

HP on day 14 and 21. In NCP, DA level was increased significantly along

with 5-HT on day 21 following icv injection of STZ. Norepinephrine was

reduced in both NCP and hippocampus on 21st day. Treatment with AChE

inhibitor in the STZ pretreated animals, compensated the STZ-induced

changes in DA and 5-HT in both regions. These data clearly indicate that the

neurochemical changes resembles the changes found in AD and the side

effects (eg. tremor etc.) observed following anticholinesterase therapy in AD

are because of altered levels of biogenic amines. This model thus can be

considered as a useful tool to investigate biochemical mechanisms involved

in AD.

Acknowledgments: HM received SRF from CSIR, Government of India.

Keywords: neurodegenerative diseases – Alzheimer’s, aging, streptozotocin,

cognition.

P21-10Apolipoprotein E gene polymorphism in Indian patientswith Alzheimer’s disease and vascular dementiaM. Tripathi

All India Institute of Medical Sciences, India

Introduction: The association of apolipoprotein E gene polymorphisms with

Alzheimer’s disease and vascular dementia has been reported in several

populations including one from a rural community in North India. However,

the association of Apo E polymorphism with VaD is yet to be established in

this population.

Material and methods: In a case–control study involving 74 cases of

dementia (AD-40 and VaD-34) and 76 age matched healthy controls blood

was analysed for the Apoe polymorphism.

Results: The frequency of e4 allele was significantly higher among cases of

AD and VaD compared with controls.

Conclusions: Our results suggests that the increased risk of developing AD

or VaD is similar among Asian Indians in presence of apo e4 compared with

Caucasian population.

Keywords: Alzheimer’s, apolipoprotein, dementia, neurodegenarative,

vascular.

P21-11O-GlcNAcylation regulates phosphorylation of tauF. Liu, L. Zhu, W. Qian, K. Iqbal, I. Grundke-Iqbal and C.-X. Gong

New York State Institute for Basic Research in Developmental

Disability, New York, Staten Island, NY, USA

Microtuble-associated protein tau is a phosphoprotein the biological activity

and cellular processing of which are regulated by the degree of its

phosphorylation. The hydroxyl groups of serine/threonine residues of tau

are also modified post-translationally by O-linked N-acetyl-glucosamine, a

novel type of O-glycosylation called O-GlcNAcylation. Because

O-GlcNAcylation and phosphorylation can act at the same hydroxyl groups

of tau, we studied the effect of O-GlcNAcylation on phosphorylation of tau

in metabolically competent rat brain slices and adult mice with or without

food depletion for 48 h. After treatment of cultured cells and brain slices with

PUGNAc that specifically elevates protein O-GlcNAcylation, we observed a

decrease in the tau hyperphosphorylation induced by okadaic acid at Ser199,

Thr212, Thr217, Ser262, Ser396, and Ser422 by quantitative Western Blots

developed with phosphorylation-dependent and site-specific tau antibodies.

Similar effect of O-GlcNAcylation on tau phosphorylation was observed in

mice. We found a decrease in protein O-GlcNAcylation and a concurrent

increase in phosphorylation of tau at above phosphorylation sites in brains of

mice after starvation for 48 h. These results suggest that tau phosphorylation

is regulated by O-GlcNAcylation and open a new avenue to study the role of

the latter in the abnormal hyperphosphorylation of tau in Alzheimer’s disease

and related tauopathies.

Keywords: O-GlcNAcylation, phosphorylation, tau.

P21-12The AICD Of APLP-2 translocalizes into the nucleus withFe65 and exerts cytotoxicity by inducing GSK-3betaexpressionK. Y. Shin, Y. Xu, C. H. Park and Y.-H. Suh




Seoul, Korea

The APP Intracellular Domain (AICD) has been shown to be involved in

gene expression in combination with Fe65. Amyloid Precursor Like Protein-

1 and -2 (APLP1 and APLP2) are known to be processed by gamma-,

epsilon-secretases and caspase-3 to generate C-terminal fragments, C57 or

C59, C50 and C31, respectively. Recently, it has been reported that the

intracellular domains of APLP1 and APLP2 are able to enhance Fe65-

dependent gene activation, similar to what has been reported for AICD. Here

we first demonstrate that APLP2-CTFs (C57, C50 and C31) translocalize into

the nucleus by Fe65-dependent mechanisms and that they form a ternary

complex with Fe65 and CP2/LSF/LBP1 transcription factor in the nucleus,

inducing glycogen synthase kinase-3beta (GSK-3beta), and tau phosphoryla-

tion and apoptosis. Previously, we reported that AICD induced GSK-3beta

expression in neuronal cells. The similar results that AICD and APLP2-CTFs

translocalize into the nucleus along with Fe65 and induce GSK-3beta

expression, leading to tau phosphorylation, might suggest that the functional

redundancy exists between them and they co-contribute to the pathogenesis

of AD.

Keywords: C-terminal fragments of APLP2, cytotoxicity, Fe65, GSK-3beta,

tau.


P21-13Role of alpha-synuclein in induction of CD44 geneexpression in human lymphocytesS.-H. Cho, S. Kim, J. K. Shin, P. S. Im and Y.-H. Suh




Seoul, Korea

Alpha-synuclein is a ubquitous 140-amino acid protein about 18 kDa. It is

the precursor protein of nonamyloid betacomponent of senile plaque (NACP)

in Alzheimer’s disease. This protein is abundant in neurons, especially

enriched in presynaptic terminals and is also found to be the major protein

component in the Lew bodies (LB), the hallmark lesions of Parkinson’s

disease, In inherited familial Parkinson’s disease, two different missense

mutations (Ala30Pro and Ala53T) has been reported which have been known

to enhance the formation of protofibrils. Recently, we presented the increased

expression patterns of it in human lymphocytes of Parkinson’s disease and

those expressions were closely correlated with dexamethasone-sensitive

apoptosis. In this study, we demonstrate that alpha-synuclein overexpression

induces the CD44 expression on human lymphocytes. CD44 is a multi-

functional cell surface glycoprotein that participates in cell adhesion during

neurite outgrowth, leukocyte homing, and tumor metastasis. Those induction

of CD44 expression on lymphocytes were greater in mutant forms of alpha-

synuclein than wild type. Especially, the overexpression of alpha-synuclein

in THP-1, a human promonocytic cell line, produced interleukin-1beta and

then as a autocrine pattern, CD44 was induced on it. Upon demonstration of

alpha-synuclein dependent CD44 generation, we further found that the

stimulation of CD44 triggered intracellular alpha-synuclein expression,

reversely. These observations shed a new light on the functional link between

alpha-synuclein and CD44.

Keywords: alpha synuclein, CD44, interleukin-1beta, lymphocyte,

Parkinson’s disease.

P21-14C-terminal fragments of APP (AICD) and Swedish APPmutation induce abnormal expression of cell cycle-related proteinsK.-W. Ahn, H.-S. Kim, C. H. Park, S. Kim and Y.-H. Suh




Seoul, South Korea

It has been reported that cell cycle-related proteins such as cyclin B1, cyclin

D1 and PCNA (proliferating cell nuclear antigen) are increased in some

specific regions of Alzheimer’s disease (AD) brains. And also hyperpho-

sphorylated tau, major component of neurofibrillary tangles which is one of

the major characteristics of AD brains, has been detected near this region. In

this study, we investigated whether C-terminal fragments of APP (AICD;

APP Intracellular Domain, C59) and Swedish mutation form of APP exerted

neutotoxicity by cell-cycle dependent mechanisms. We found that expression

of AICD or Swe-APP into NGF-differentiated PC 12 cells or rat primary

cortical neurons increased mRNA and protein levels of cyclin B1, cyclin D1.

In addition, p21, prolyl isomerase 1, PCNA protein levels was also

upregulated in the neuronal cells. These results suggest that some

disturbances in cell cycle regulation may be involved in the APP-CTs or

Swe-APP induced neurotoxicity and this might contribute to AD pathogen-

esis.

Keywords: AD pathogenesis, APP, cell cycle protein, hyperphosphorylated

tau, Swedish mutation.

P21-15Dehydroevodiamine hydrogen chloride extracted fromEvodia Rutaecarpa Bentham prevents impairment oflearning and memory and neuronal lossC. H. Park*, S. H. Choi*, S. H. Lee�, H. S. Kim* and Y.-H.Suh*

*Department of Pharmacology, College of Medicine; �Departmentof Neurosurgery, Nat’l Creative Res. Initiative Center for

Alzheimer’s Dementia and Neuroscience Research Institute, MRC,

Seoul Nat’l University, Seoul, South Korea

We found that dehydroevodiamine hydrogen chloride (DHED), which was

purified from Evodia Rutaecarpa Bentham, had anticholinesterase effect and

its beneficial effects on cognitive dysfunction and neuronal damage in the

animal models related to Alzheimer’s disease. We further investigated the

effects of DHED on neurotoxicities induced by beta amyloid (Ab), a

recombinant carboxyl-terminal 105 amino acid fragment (CT105) of amyloid

precursor protein, H2O2 and glutamate (Glu) in primary cultured neurons of

rat. We also assayed the effects of DHED on NMDA receptor. The

pretreatment of DHED (0.5 4 luM) significantly reduced cell death induced

by Ab(10 uM), CT105 (10 uM), H2O2 (150 uM) and Glu (1 mM). DHED

inhibited NMDA-induced whole cell ionic currents (19.5 ± 3.9% at 100 uM)

and antagonized the binding of MK-801 to NMDA receptor (IC50;

32.9 uM). The cognitive dysfunction induced by an injection of CT105

(6 nmol) into rat hippocampus was significantly improved by the adminis-

trations of DHED (10 mg/kg, p.o. daily) for 3 weeks in the water maze test.

DHED reduced immunoreactivities of cyclooxygenase-2 and inducible nitric

oxide, and the neuronal loss in the hippocampus of the rats. These results

show that DHED might attenuate neuronal loss and cognitive dysfunction

caused by brain damages and toxic metabolites of APP through multi-

neuroprotective mechanisms antagonizing oxidative stress, inflammatory

response and NMDA receptor. Thus DHED has potential therapeutic effects

on not only AD type dementia but also vascular dementia and stroke.

Keywords: neurodegenerative diseases – Alzheimer’s, neuroprotection,

neurotoxicity.

P21-16Lack of early activation of endoplasmic reticulum stressin beta-amyloid neurotoxicityM. S. Yu, K. C. Suen, N. S. Kwok, K. F. So and R. C. C. Chang

Laboratory of Neurodegenerative Diseases, Department of

Anatomy, Faculty of Medicine, and Central Laboratory of the

Institute of Molecular Technology for Drugs Discovery and

Synthesis, The University of Hong Kong, Pokfulam, Hong Kong

Beta-amyloid (Ab) peptide neurotoxicity plays significant roles in Alzhei-

mer’s disease (AD). Mutation of presenilin-1 (PS-1) gene sensitizes neurons

to Ab toxicity, also, causes dysfunctions of endoplasmic reticulum (ER).

Therefore, ER stress receives increasing attention to AD. This study aimed to

investigate different signaling pathways for ER stress responses in Abneurotoxicity. Rat cortical neurons exposed to Ab peptide did not showed

induction of alternative splicing in Xbp-1 mRNA by IRE-1, up-regulation of

Xbp-1 mRNA by ATF-6 and PERK phosphorylation. After 16-h treatment of

Ab peptide, there were induction of GRP 78 and GADD153 as well as

activation of caspase-12 and -7. These data suggested that ER stress is not an

early event involved in Ab neurotoxicity. Activation of JNK and

phosphorylation of eIF2a were found at early time-point, however, these

signaling events and neuronal cell death were not mediated by ER stress

responses. Taken together, ER stress seems to be a late response in Abpeptide-induced toxicity.

Keywords: Alzheimer’s disease, amyloid, ER stress, neurotoxicity, PERK.

52 � 2004 International Society for Neurochemistry, Journal of Neurochemistry, 88 (Suppl. 1)

P21-17Effects of nerve growth factor on beta-amyloid25-35-induced neuronal injuryT. Huidong, C. Xuehua and C. Shengdi

Department of Neurology, Rui Jin Hospital, Shanghai Second

Medical University, Shanghai, China

Objective: To investigate the protective and therapeutic effects of nerve

growth factor (NGF) on beta-amyloid25-35-induced hippocampal neuron

injury.

Methods: Hippocampal neurons were cultured in neural basal med-

ium + B27 factor for 7 days, then divided into NGF and control groups.

Beta-amyloid25-35 (10 lg/ml) was added before and after addition of

NGF(10 lg/ml) and culture fluid separately in each group. The integrity of

hippocampal neurons were evaluated by beta-tublin & MAP2 immunohis-

tochemistry. Cell viability and ChAT-positive neurons were determined by

MTT and ChAT immunohistochemistry.

Results: Beta-tublin & MAP2 immunohistochemistry demonstrated the

integrated hippocampal cells. MTT and ChAT immunohistochemistry

showed that the cell viability and the number of ChAT-positive neurons

were decreased significantly. The obvious protective and mild therapeutic

effects of NGF on beta-amyloid25-35-induced hippocampal neurons injury

was found.

Conclusions: NGF can effectively prevent and mildly treat the injury of

hippocampal neurons induced by beta-amyloid25-35. It indicates that NGF

may have protective and therapeutic effects for Alzheimer’s disease.

Keywords: Alzheimer’s disease, beta-amyloid, cell injury, nerve growth

factor.

P21-186-Hydroxymelatonin protects against oxidativeneurotoxicityD. S. Maharaj, B. D. Glass and S. Daya

Division of Pharmacology, Faculty of Pharmacy, Rhodes

University, Grahamstown, South Africa and School of Pharmacy

and Molecular Sciences, James Cook University, Townsville, QLD,

Australia

The brain is particularly vulnerable to free-radical induced damage,

especially if one considers the large amount of oxygen utilization by the

brain. Free radical damage has long been suspected to play a role in the

progression of various neurological conditions. Thus, the use of antioxidants

to combat this free radical production in the brain is becoming increasingly

popular. Its major hepatic metabolite and photoproduct 6-hydroxymelatonin

(6-OHM) shares this property. Because of the implication of singlet oxygen

and quinolinic acid (QA) in neurotoxicity, the objective of this study was to

investigate the ability of to 6-OHM to scavenge singlet oxygen and evaluate

the ability 6-OHM to scavenge superoxide anions and reduce QA-induced

neurotoxicity in the hippocampus in vivo. Furthermore, the effect of 6-OHM

against QA induction of heat shock protein 70 (Hsp70) and apoptosis in the

rat hippocampus was determined using western blotting and immunohis-

tochemistry, respectively. The results show that 6-OHM is an efficient

inhibitor of singlet oxygen formation as indicated by the rate constants and

quantum yields reported for 6-OHM and zinc phthalocyanine (ZnPc)

respectively. 6-Hydroxymelatonin, appears to reduce QA-induced superoxide

anion generation in the hippocampus, providing evidence of the neuropro-

tective effects of 6-OHM. 6-OHM was also shown to reduce QA expression

of Hsp70 and programmed cell death in the hippocampus. The results of the

present study indicate that this agent has a definite role to play as a

neuroprotective agent.

Keywords: apoptosis, Hsp70, neurodegeneration, neuroprotection, oxidative

stress.

P21-19Calcium in neurodegenerative disorders – a holisticapproachA. Palotas, B. Penke, L. Kemeny, Z. Janka, G. Laskay and

J. Kalman

University of Szeged, Szeged, Hungary

Introduction: Efforts to elucidate the pathomechanism of beta-amyloid in

Alzheimer’s disease and other factors in diverse neurodegenerative disorders

have yielded an increasing pile of hypotheses. When analyzing thousands of

scientific papers, the involvement of the central secondary messenger,

calcium, becomes apparent.

Methods: Resting intracellular calcium concentration of neurons, glias,

fibroblasts and lymphocytes were assessed utilizing comparative fluorimetric

methods with or without treatment of cultures with beta-amyloid. Medline

search was performed to supplement and justify the involvement of calcium

in various neurodegenerative disorders.

Results: Disturbed calcium homeostasis is present in all cell-types examined

after beta-amyloid treatment. Medline-search points out the role of calcium

disregulation in several neurometabolic disorders, including schizophrenia,

Parkinson’s, Huntington’s, amyotropic lateral sclerosis, etc.

Discussion: Our results and data from Medline-search confirm that calcium

imbalance might be a common underlying factor in brain pathologies.

Disturbed calcium interferes with some of the many biochemical pathways

characteristic of a certain disorder, determined by environmental and genetic

factors, yielding disease-specific pathologies. By targeting calcium, this new

information promises to broaden our understanding of health and illness and

the approaches we take to treating disease.

Keywords: aging, Alzheimer’s disease, calcium, neurodegenerative dis-

eases, second messengers.


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Neurodegenerative diseases: Alzheimer's disease