Poster Presentations P2 S411

was GSK3b and CDK-5. The pineal and retinal melatonin regulates endog-

enous circadian rhythms, and has various physiological functions including

neuromodulatory and vasoactive actions, antioxidative and neuroprotective

properties. Our recent studies have demonstrated that melatonin efficiently

attenuates Alzheimer-like tau hyperphosphorylation in Tg2576. Methods:

Four-month-old (n ¼ 4-5)and Eight-month-old (n ¼ 4-5) Tg2576 animals

(Tgs) were daily injections of melatonin (14mg/kg) for 4 months. Respective

controls included non-transgenic littermates (Lts) (n ¼ 4-5), and untreated

Tgs (n ¼ 4-5). After 4 months of treatment, Hyperphosphorylated tau,

GSK-3b and CDK5 were determined by Western blotting, immunohisto-

chemistry with specific antibodies. Results: The long-term influence of mel-

atonin on behavior, biochemical and neuropathologic changes in Tg2576. In

Eight-month-old mice, Hyperphosphorylated tau epitopes were substantially

decreased as assessed with the pT205 and pS404 antibodies in mel-treated Tg

mice. The untreated Tg mice show increased levels of tau hyperphosphory-

lation and increased activated CDK5. In twelve-month-old mice, Hyperphos-

phorylated tau were substantially decreased as assessed with the pT231

antibodies in mel-treated Tg mice. The untreated Tg mice show, together

with increased amyloidogenesis, increased levels of tau hyperphosphoryla-

tion and increased activated GSK-3b. Four months of Melatonin treatment

reduced the burden of amyloid plaques and the levels of hyperphosphory-

lated tau. Melatonin reduced the activated CDK5 and GSK-3b respectivly

in 8 months and 12 months. Conclusions: Melatonin can exert multiple pro-

tective effects on both amyloidogenesis and tau hyperphosphorylation via

regulate the activated CDK5 and GSK-3b in transgenic mice Tg2576 of dif-

ferent ages.

P2-333 ENHANCED SPONTANEOUS RELEASE OF

NEUROTRANSMITTER UNDERLIES EARLY

CHANGES IN SYNAPTIC FUNCTION BY BETA-

AMYLOID

Mauro Fa1, Elena Leznik1, Ipe Ninan2, Fabrizio Trinchese1, Shumin Liu1,

Ian J. Orozco1, Peter Koppensteiner3, Ottavio Arancio1, 1Columbia

University, Taub Institute, New York, NY, USA; 2New York

University, Department of Psychiatry, New York, NY, USA; 3Universitat

Wien, Diploma Programme Molecular Biology, Wien, Austria.Contact e-mail: mf2257@columbia.edu

Background: Several lines of evidence suggest that Alzheimer’s disease

(AD) begins as a synaptic disorder leading to cognitive deficits. In mice,

nanomolar concentrations of oligomeric b-amyloid peptide (Ab) cause a re-

duction in hippocampal synaptic plasticity and memory associated with

a decrease of both the frequency and the amplitude of the spontaneously

occurring release of neurotransmitter from the presynaptic terminal in the

absence of the action potential (mEPSC). A brief application of picomolar

Ab42 concentrations, in turn, enhances hippocampal synaptic plasticity and

memory. To examine the phenomena occurring during the transition from

positive to detrimental effect of Ab, we exposed both primary neuronal cul-

tures and acute hippocampal slices from adult mice to picomolar Ab42 for

prolonged time. Methods: Primary neuronal cultures and slices from the

hippocampus were used in our experiments. Neurons were recorded by us-

ing both patch clamp technique and extracellular recording methods. Imag-

ing technique with FM dye was used to investigate release of

neurotransmitter from synaptic boutons in cultures. Distribution of presyn-

aptic proteins, synaptophysin and synapsin I, and post-synaptic protein

GLUR1 was investigated through immunocytochemistry. Results:

Twenty-four hour exposure to low picomolar concentrations of Ab42 in-

creased the mEPSC frequency (without affecting their amplitude), the num-

ber of active presynaptic release sites and clusters for the pre- and post-

synaptic proteins while it impaired synaptic plasticity. In accordance

with results from the primary culture neurons, one hour exposure to low

picomolar Ab42 in acute hippocampal slices produced a delayed increase

in the frequency of mEPSCs and reduced long-term potentiation (LTP)

in the CA1 region of the hippocampus. Conclusions: This body of evi-

dence strongly supports the hypothesis that the earliest amnesic symptoms

of AD occurring in the absence of any other clinical signs of brain injury

involve an increase of neurotransmitter release from the presynaptic termi-

nal due to Ab.

P2-334 THE SYNAPTIC MECHANISMS OF GLYCOGEN

SYNATHESE KINASE-3BETA IN REGULATING

LEARNING AND MEMORY

Jian-Zhi Wang, Ling-Qiang Zhu, Dan Liu, Juan Hu, Qun Wang,

Tongji Medical College, Wuhan, China. Contact e-mail: wangjz@mails.tjmu.edu.cn

Background: Previous studies have demonstrated that upregulation of gly-

cogen synathese kinase-3beta (GSK-3beta) impairs learning and memory in

rats and transgenic mouse models, and ctivation of GSK-3beta inhibits long

term potentiation with mechanisms involving a decreased presynaptic glu-

tamate release. Methods: Wild type and dominant negative GSK-3 plas-

mids were transfected to regulate the activity of GSK-3. FM4-64

releasing by time lapse with confocal microscopy in primary hippocampus

neurons and pHluorin-VAMP2 fluorescence detecting in Neuro2A cells by

total internal reflecting fluorescence microscopy (TIRFM) were used to

evaluate the exocytosis stimulated by 90mM K+, Fluorescence resonance

energy transfer (FRET) and immunoprecipitation in synaptosome were

used to assay the formation of SNARE complex, which play an important

role in exocytosis. GSK-3 activity was determined by 32P-labelling assay.

Results: We have recently demonstrated that activation of GSK-3beta

could inhibit calcium influx through phosphorylating the intracellular

loop connecting domains II and III (LII-III) of P/Q-type Ca2+ channels,

which leads to decrease of intracellular Ca2+ influx through the P/Q-type

voltage-dependent calcium channel (VDCC). GSK-3beta interferes with

the formation of SNARE complex through (i) weakening the association

of synaptobrevin with SNAP25 and syntaxin; (ii) reducing the interaction

between the phosphorylated LII-III and synaptotagmin, SNAP25 and syn-

taxin; and (iii) inhibiting dissociation of synaptobrevin from synaptophysin

I, all of which are required for SNARE complex formation and thus for an

efficient exocytosis. Conclusions: These results suggest that GSK-3beta

regulates synaptic vesicle cycle by affecting multiple steps in vesicular exo-

cytosis

P2-335 NEURONAL SIGNALING MECHANISMS

UNDERLYING SYNAPTIC DEFICITS IN

ALZHEIMER’S

Grace E. Stutzmann, Shreaya Chakroborty, Ivan Goussakov,

Megan Miller, Rosalind Franklin University/The Chicago Medical School,

North Chicago, IL, USA. Contact e-mail: Grace.Stutzmann@

rosalindfranklin.edu

Background: The devastating cognitive deficits that define Alzheimer’s

disease are associated with structural and functional impairments in neu-

ronal synapses. This breakdown of a critical neuronal signaling site pre-

cedes the formation of beta amyloid plaques and neurofibrillary tangles,

and correlates with the degree of memory loss. Yet, it is unclear what

pathogenic mechanism is contributing to alterations in synaptic integrity.

A possible culprit may be alterations in dendritic calcium signaling, which

can result in both synaptic transmission alterations and structural abnor-

malities of synapses. Methods: We use video-rate 2-photon imaging cou-

pled with whole cell patch clamp recording from cortical and hippocampal

pyramidal neurons to simultaneously record calcium signals from dendritic

compartments and synaptic responses from control and AD mouse

models. In addition, hippocampal synaptic plasticity studies are conducted

using field potential and sharp electrode recordings, while RTPCR and im-

munoblotting approaches identify changes in calcium signaling and synap-

tic factors at the mRNA and protein level. Results: Focusing on dendritic

processes and spines, we show that in the AD mice there are profound

increases in ER calcium release mediated through ryanodine receptors.