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Folia Neuropathologica
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vol. 43

Original article
Alzheimer\'s disease related peptides affected cholinergic receptor mediated poly(ADP-ribose) polymerase activity in the hippocampus

Agata Adamczyk
Henryk Jęśko
Robert Piotr Strosznajder

Folia Neuropathol 2005; 43 (3): 139-142
Online publish date: 2005/09/30
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Communicating author:
Agata Adamczyk, Department of Cellular Signaling, Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego St, 02-106 Warsaw, Poland, tel. /fax +48 22 608 65 72, e-mail: agatazambrzycka@hotmail.com

Our previous study presented that activation of the muscarinic cholinergic receptor (mChR) by non hydrolysable analog of acetylcholine, carbachol and activator of G protein, GTP(g)S significantly enhanced inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) formation and that this signaling events are significantly altered by amyloid beta peptide (Aβ) [27]. In this study, the mChR evoked signals were investigated on the level of DNA-bound enzyme poly(ADP-ribose) polymerase (PARP-1, EC The recent data suggested that PARP-1 is the new nuclear target for signal transduction processes evoked by receptor(s) stimulation or membrane depolarization [12,21,23]. Activated PARP-1 cleaves NAD into nicotinamide and forms long and branched polymers of ADP-ribose on over forty nuclear proteins and
PARP-1 itself. Poly (ADP-ribosyl) ation has been implicated in the regulation of a diverse array of cellular processes ranging from DNA repair, chromatin organization, transcription, to replication [2,7,9,15,21,25]. However, oxidative stress-induced DNA strand breaks that stimulates PARP-1 lead to bNAD+ and ATP depletion and to necrotic or apoptotic processes [10,13,26]. The pathophysiological significance of PARP-1 over-activation is suggested in brain ischemia, diabetes, inflammation and cancer [4,11,21]. Increased expression of this enzyme and accumulation of poly(ADP-ribose) was observed in Alzheimer's and Parkinson's brain [14,17,19]. Our resent data indicated the involvement of mChR
in the regulation of PARP-1 activity through IP3 receptor pathway [23]. In the present study, we investigated the effect of Alzheimer's disease (AD) related Aβ and NAC peptide on mChR-dependent signal transduction to PARP-1.

Material and methods

Male Wistar rats, 4-month-old (250-300 g), were supplied from the Animal Breeding House of the Medical Research Centre (Warsaw). The Institutional Ethics Committee accepted the research project.

Adenine[C14]NAD (sp. activity: 252 mCi/mmol), was obtained from Amersham, Buckinghamshire, UK, Aβ 25-35, carbachol, GTPgS, and the all other reagents were from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Preparation of Aβ and NAC peptides
Aβ 25-35 was dissolved in bidistilled deionized (BDD) water at 2.5 mM concentration and incubated at room temperature to obtain the aggregated form as described previously [20]. Aβ was used at 25 µM final concentration. NAC peptide was stored lyophilized, and 100 µM stock solution was prepared in BDD water and then used at final 10 µM concentration. To obtain the aggregated form NAC was incubated for 3 days at 37°C.

Preparation of brain slices
Animals were killed by decapitation, the brain was quickly removed, hippocampi were dissected and cross-chopped into slices (350x350 µm) using a McIlwain tissue chopper. The slices were placed in ice-cold Krebs buffer (in mM: NaCl 124, KCl 5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 26 and glucose 10) equilibrated with 5% CO2 in 95% O2 to maintain a pH of 7.4 and pre-incubated for 30 min at 37°C in Krebs buffer. Then, CaCl2 was added to final 2 mM concentration and incubation was prolonged for 30 min at 37°C in the presence or absence of the following compounds: Aβ 25-35 (25 µM), NAC (10 µM), carbachol (1 mM) together with GTP(g)S (100 µM), TMB-8 (10 µM). After incubation, the slices were homogenized in glass-glass homogenizer and used for determination of the PARP-1 activity.

Determination of the PARP-1 activity
The PARP-1 activity was assayed as described previously [22]. The total reaction mixture in the final volume of 100 µl contained 400µM [adenine-14C]NAD as a substrate (2x105 d.p.m., sp. activity: 252 mCi/mmol), 100 mM Tris-HCl buffer pH 8.0, 10 mM MgCl2, 1mM dithiothreitol (DTT) and 200 µg of protein. The reaction was carried out for 1 min at 37°C and stopped with 800 µl of ice-cold 25% trichloroacetic acid (TCA). The precipitate was collected on Whatman GF/B filters, washed five times with ice-cold 5% TCA and processed for determination of radioactivity in Bray's scintillation fluid using scintilator counter Wallac 1409 LKB.
In the present study, we examined the role of mChR on PARP-1 activation in the brain cortex and hippocampus. Moreover, the effect of both AD related peptide Aβ and NAC on this receptor mediated signaling to PARP-1 was evaluated. The data showed that carbachol at 1mM concentration together with GTP(g)S at 100 µM significantly enhanced the PARP-1 activity by about 100% in the hippocampus with no effect on this enzyme in the brain cortex (Fig. 1). Inhibition of IP3 receptor through TMB-8 (10 µM) decreased PARP-1 activation evoked by carbachol/GTP(g)S in the hippocampus (Table I). MChR stimulation by carbachol/GTP(g)S had no effect on free radicals generation and macromolecules oxidation (data not shown). In addition, this receptor stimulation had no effect on the PARP-1 activity in the presence of Aβ and NAC peptide (Fig. 2). However, both peptides, Aβ and NAC significantly stimulated this enzyme by 88% and 23%, respectively by free radicals evoked DNA strand breaks (Fig. 2).

The major finding of this study is that AD related peptide Aβ and NAC disturbed phosphoinositide signaling to PARP-1 in the hippocampus. These and our recent results presented PARP-1 as a nuclear target for mChR pathway [23] are in agreement with the previous data of Homburg et al [12]. They showed that depolarisation of neurons activated PARP via
IP3-induced Ca2+ signaling. In addition, it was shown that N-methyl-D-aspartate (NMDA) receptors stimulation could mediate PARP-1 activation [18,22]. Moreover, the present data indicated that Aβ and NAC peptide enhanced basal PARP-1 activity. These results supported our earlier data that both peptides induced reactive oxygen species (ROS) generation and DNA degradation [1]. It is well documented that PARP-1 is the earliest and the most sensitive indicator of DNA strand breaks evoked by oxidative stress [8,28]. Over-activation of PARP-1 leads to cellular bNAD+ depletion, apoptotic inducing factor (AIF) release and cell death [13,26]. Aβ together with NAC peptide is a major component of senile plaques in AD and through ROS generation could participate in neuronal cell death [3,5,6,16,24]. Our previous results showed that mChR-dependent signal transduction in the rat brain is damaged by Aβ peptide that significantly inhibited phosphatidylinositol-4,5-bisphosphate phospholipase C (PIP2-PLC) activity through ROS formation and membrane lipids peroxidation [20,27]. The present data indicated that Aβ and NAC peptide decreased PARP-1 activation evoked by mChR stimulation. We therefore consider that both peptides through the free radicals mechanism inhibited PIP2-PLC activity and IP3 liberation and disturbed signal transduction from mChR to PARP-1.

This work was supported by the Grant PBZ-MIN-001/P05/16 and by statutory budget of the Polish Academy of Science Medical Research Center (Theme No 1).

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Copyright: © 2005 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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