INTRODUCTION

Alzheimers disease (AD) is characterized by regional neurodegeneration, synaptic loss and the presence of senile plaques (SantaCruz et al. 2005). The senile plaques consist of abnormal interneu-ronal growth, dead cells and beta amyloid. There has been increasing evi­dence which suggests that the buildup and ag­gregation of beta amyloid peptide (Aβ) in the brain plays a primary role in the pa­thogenesis of AD (Piccini et al. 2005).

 Although Aβ has been demonstrated to be neurotoxic in cell culture (Butterfield et al. 2004), the mechanism by which it ex­erts its toxicity is still unclear. Several mechanisms have been suggested in­cluding increased sensitivity to excito­toxins, alterations to calcium homeosta­sis (Celsi et al. 2006), activation of re­ceptor that precipitates cell destruction and activation of inflammatory pathways (Zou et al. 2006). 

 Recently, the involvement of oxidative stress in the pathogenesis of AD has been suggested (Wang et al. 2008). The AD brain was reported to have increased lipid peroxidation, increased carbonyl modification of proteins and increased oxidation of mitochondrial DNA (Arimura& Kaibuchi 2005).

 Increase oxidative stress occurs when the balance between free radical genera­tion and antioxidant capacities shifts to­ward free radical generation leading to oxidative damage to lipids, protein, RNA and DNA. This is not surprising as the brain is more susceptible to oxidative damage compared to other organs or tissues, due to its high rate of oxygen consumption, high polyunsaturated lipid content, and relative paucity of antioxi­dant enzymes (Puttfarcken 1996).

Understanding how Aβ acts and its probable association to oxidative stress is important for the formulation of treat­ment and preventive steps against neu­rodegeneration. Therefore, in this study, the involvement of oxidative damage in the mechanism of Aβ-toxicity resulting in neuronal cell death was determined by measuring oxidative DNA damage. The involvement of oxidative damage was further elucidated by determining the ef­fect of TRF.

MATERIALS AND METHODS

SH-SY5Y neuroblastoma cell culture

Human neuroblastoma SH-SY5Y cells were gifts from Dr. Coral Sanfeliu of the Institut d’Investigacions Biomèdiques de Barcelona, Spain. Cells were grown un­der the following condition: 50% of EMEM with Earles Salt pH 7.2 (Flowlab, Australia) and 50% Ham's F-12 pH 7.2 (Sigma, USA) supplemented with 200mM Non-essential amino acid (NEAA) (Flowlab, Australia), 10mg/ml gentamicin (PAA, Austria) and 10% Foetal Bovine Serum (FBS), heat inactivated (PAA, Austria). Cells were subcultured after wash with phosphate buffered saline (PBS) and trypsin. Cells were then seeded at 1.5 x 104 cells per ml on 96-well plates. Cultures were maintained in 5% CO2 / 95% air at 37OC. Experiments were carried out on the 7th-9th day.

Preparation of Aggregated Aβ

The stock solution of Aβ1-42 (Calbiochem, German) was dissolved at 1mg/ml in 50Mm Tris-HCL, pH ≥ 9.0. The peptide was diluted to 20μΜ with calcium, mag­nesium-free PBS and stored at -20oC. The stock solution was then diluted to a final concentration of 10μΜ followed by incubation at 37oC for 24 hours before use.

Experimental Design

Cells were divided into 4 groups: un­treated cells served as control, treated with 10μM Aß peptide for 24 hours, 5mg/ml TRF for 24 hours followed by 10μM Aß peptide for 24 hours (pre treatment) or 10μM Aß peptide for 24 hours followed by 5mg/ml TRF for 24 hours (post treatment). All cultures were maintained in 5% CO2 / 95% air at 37oC. TRF was pur­chased from Golden Hope Biorganic Sdn. Bhd. (Malaysia). DNA damage, cell viability and apoptosis were determined by using MTS assay, comet assay and Propidium Iodide & Calcein-AM staining respec­tively.

Comet assay

DNA damage was determined by using the Comet assay according to the me­thod described by Singh et al. (1988), with minor modifications. 105μl of normal melting agarose (NMA) was added to frosted microscope slides (Curtin Matherson, USA), covered with cover­slips and kept for 10 minutes to solidify. Co­verslips were removed and 20μl cells with 80μl low melting point agarose (LMA) were added to the slides (Sigma, USA). The slides were then immersed in a jar containing cold lysing solution (2.5 M NaCl, 100 mM ethylene-diaminetetra-acetic acid, 10 mM Tris at pH 10, 1% Triton X-100, 1% sodium N-lauroyl sar­cocinate, 10% dimethylsulfoxide)  for at least 1 hour. After lysis, the slides were placed in electrophoresis box filled with electrophoresis buffer (0.3 M NaOH and 1 mM ethylene-diaminetetra-acetic acid). The cells were incubated for 20 minutes to allow for unwinding of DNA and ex­pression of alkali-labile sites. All of these steps were conducted under dim light to prevent additional DNA damage. After electrophoresis, neutralization buffer (0.4M Tris, pH 7.5) was added to neu­tralize the alkali, and the slides were left at room temperature for 5 minutes. This step was repeated twice. Ethidium bro­mide (Sigma, USA) was added to each slide, covered with a co­verslip, kept in a humidified box and fi­nally analyzed using a fluorescence mi­croscope (AxioCam MRC, Carl Zeiss, Germany).

MTS assay

The MTS assay purchased from (Promega, USA), employs MTS and the electron coupling agent phenazine me­thosulphate (PMS). MTS is converted into a medium soluble forma­zan product by dehydrogenase enzymes found in metabolically active cells. 20 μl of MTS reagents was pipetted into each well containing the samples in 100μl of cul­ture medium. The plates were then incu­bated for 2 hours in 5% CO2/95% air at 37oC. The optical density (OD) of the wells was determined using a microplate reader at 490-nm wavelength (Versa max, Japan).

Propidium Iodide & Calcein-AM staining

Staining to detect cell death is based on the principle that live cells have intracel­lular esterase that converts non-fluores­cent cell-permeable calcein-AM to the intensely fluorescent calcein which is re­tained within the cells. Viable cell mem­branes are impermeable to propidium iodide. Dead cells however, allow propi­dium iodide to enter and bind to nucleic acid. Thus live cells stained green with calcein-AM while dead and apoptotic cells stained red with propidium iodide (Sigma, USA). 30μg/ml calcein-AM and 7.5 μg/ml propidium iodide were added to the cell cultures in chamber slides (Nunc, Denmark) and incubated for 30 minutes. Thereafter, cultures were washed with PBS, fixed with fresh 4% paraformalde­hyde and then incubated for 30 minutes at 37OC. Slides were washed and cover­slips mounted for microscopic examina­tion (AxioCam MRC, Carl Zeiss, Germany).

RESULTS

DNA Damage Studies

Figure 1 shows DNA damage in the treated and untreated cells as deter­mined by comet assay. Cells were grouped according to the length of comet tail (%). Cells without DNA fragmentation were classified as no DNA damage, cells with DNA fragmentation were classified as having mild damage and cells with major fragmentation as severely dam­aged. Re­sults showed that incubation with 10μM Aß peptide significantly in­creased the DNA damage (p<0.05). However, cells treated with TRF before (pre treatment) or after (post treatment) incubation with Aβ was observed to have a significantly reduced number of DNA damage. There was no significant differ­ence observed between the number and severity of DNA damage in pre and post TRF treated groups.

Determination of Aβ Peptide toxicity

The concentration of Aβ peptide which can induce 50% cell death (IC50)was determined by incubating cells with in­creasing concentration of Aβ peptide. Figure 2 shows that this concentration was 10μM and hence 10μM Aβ peptide was the concentration used in subse­quent experiments.

Neuroprotection  Studies

Figure 3 shows that 10μM Aβ signifi­cantly increased neuronal death. Pre and post treatment with 5mg/ml TRF signifi­cantly increased the number of viable cells compared to the cells treated with Aβ only and thus demonstrated that TRF was able to prevent cell death induced by Aß peptide.

Figure 4 shows the results of fluores­cence staining using calcein AM and propidium iodide. Control live neuron cells were stained green (a) whereas cells exposed to 10μM Aß peptide were stained red with (i) cell shrinkage and (ii) condensed chromatins, characteristics of apoptotic cells (b). Cells pretreated with 5mg/ml TRF before exposure to Aß un­derwent early apoptosis but still retained membrane integrity (c). TRF post treated cells showed increased number of viable cells compared to cells exposed to Aβ only demonstrating that post treatment of cells with TRF prevented cell death in­duced by Aß (d).

DISCUSSION

Recent evidence suggests that Aβ pep­tide may contribute to the progressive neuronal loss and/or may be directly neu­rotoxic both in vitro and in vivo (Pike et al. 1993). This is consistent with the present finding where Aβ peptide was observed to significantly decrease the number of viable cells (Figure 2). The induction of cell death may be elicited via apoptosis (Behl et al. 1994). The present finding (Figure 4b) showed that the num­ber of apoptotic cells was increased while the number of viable cells decreased when exposed to Aβ peptide.

 Aβ peptide may exert its cytotoxic ef­fects by activating the apoptotic pathway via free radical damage and/or induction of the signaling molecules (Pereira et al. 1999). Free radicals are constantly gen­erated in vivo and have been reported to be significant contributors to the devel­opment of chronic degenerative disorders such as Alzheimers disease (Ames et al. 1993). Although multiple antioxidant re­pair systems exist to help control this process, reactive species still cause damage to biomolecules, cells and tis­sues.

 DNA damage is probably the most sig­nificant biological target because this may ultimately lead to altered gene ex­pression, disruption of cellular repair me­chanism and distorted cellular function (Heaton et al. 2002). Thus DNA damage was measured in this study after cells were incubated with Aβ peptide with and without treatment with TRF.

 Comet assay was used to measure DNA damage and visual scoring method was applied to evaluate the amount of DNA damage (Collins 2004). The present results showed that DNA damage was significantly increased in cells incubated with Aβ peptide (Figure 1). This may be attributed to the observation that Aβ pep­tide could generate ROS which contri­bute to the DNA damage.

 Treatment with TRF, either before (pre) or after (post-treatment) incubation with Aβ peptide confirmed the neuroprotective effect of TRF. The data from this study showed that TRF prevented cell death, minimized apoptosis and reduced the DNA damage induced by Aβ peptide. Many studies have shown that vitamin E and its isomers exert neuroprotective effects against ROS-induced cell death in vitro (Sen et al. 2006; Mazlan et al. 2006). A recent report also demonstrated that AD patients with moderately severe impairment respond favorably to α-toco­pherol by slowing the progression of the disease (Sano et al. 1997). An epidemi­ological study involving 633 persons over 65 years old suggests that the use of high dose vitamin E supplements may lower the risk of AD (Morris et al. 1998). The neuroprotective effect of TRF may be exerted via its antioxidant effects or by acting at the gene level, affecting the ex­pressions of the signaling molecules of the apoptotic pathway.

 The results thus show the relationship between the oxidative DNA damage, apoptosis and cell death induced by Aβ peptide which is prevented by TRF. In conclusion, Aβ peptide cause DNA dam­age and ultimately cell death via apopto­sis probably by inducing oxidative DNA damage. This is further supported by the fact that TRF, an antioxidant is able to prevent the DNA damage, apoptosis and cell death.

ACKNOWLEDGEMENT

This study was funded by UKM-FF-03-FRGS0006-2006 research grant.