Telencephalin protects Paju cells from beta-amyloid protein-induced apoptosis☆

Heping Yang , Dapeng Wu Xiaojie Zhang, Xiang Wang Yi Peng Zhiping Hu

1Department of Neurology, Shangrao No.5 People’s Hospital, the Second Affiliated Hospital of Branch to Nanchang University, Shangrao

334000, Jiangxi Province, China

2Department of Neurology, Xiangya Second Hospital, Central South University, Changsha 410011, Hunan Province, China

3Department of Neurology, Ruikang Hospital Affiliated to Guangxi Traditional Chinese Medical University, Nanning 530011, Guangxi Zhuang Autonomous Region, China

4Department of Psychiatry, Xiangya Second Hospital, Central South University, Changsha 410011, Hunan Province, China

INTRODUCTION

Beta-amyloid protein (Aβ) accumulation initiates a cascade of pathological events in Alzheimer’s disease (AD), resulting in the formation of senile plaques[1], as well as neural damage and death[2-3].

Neuroprotective therapy is a promising method for treating AD, which prolongs neuronal survival.However, the endogenous factors required to induce a neuroprotective effect in neurons remains unknown.

Telencephalin/intercellular adhesion molecule-5 (TLN/ICAM-5) is an adhesion molecule in the adult mammalian brain that is expressed exclusively in the somatodendritic membranes of telencephalic neurons[4-6].TLN participates in immunoregulation in the brain and promotes growth of neuron dendrites[7].Moreover, it reduces the damage caused by Aβ in Paju cells[8], indicating that TLN may protect neurons from Aβ toxicity.However,few studies have investigated the protective effects of TLN on Aβ-induced neuronal apoptosis.

We reported previously that TLN/ICAM-5 attenuates axonal disruption induced by Aβ35and the 42 amino acid long β amyloid peptide (Aβ42) in Paju cells[8].Therefore, this study investigated the later events of neuronal degeneration induced by Aβ42in the differentiated Paju cell line and the neuroprotective effects of TLN.

RESULTS

Paju-TLN cell morphology

After 24 hours in 1.0 nmol/L Aβ treated culture medium, thin processes and small bodies of Paju-neo cells were visible, but no remarkable changes were observed in Paju-TLN cells.At 48 hours, a small quantity of Paju-neo cells floated; some cells were contracted, round and showed light refraction.Paju-TLN cells showed thin processes and small cell bodies.At 72 hours, many Paju-neo cells were floating and exhibited vacuoles and particles in adherent cells.Paju-TLN cells were clear,contracted, and round.Most Paju-neo cells were dead by 96 hours and were floating,and some were colorless; living cells presented with an incomplete structure.Most Paju-TLN processes became thin, and cell bodies became small.Some floating cells were present (Figure 1).After 96 hours,Paju-neo cells disappeared.At day 5, all Paju-TLN cells died.

Survival rate of Paju cells

The survival rates of Paju-neo and -TLN cells decreased over time, with significant differences at 24 hours (Paju-TLN=64.31 ±5.45%, Paju-NEO=53.67±6.89%,P＜0.05).At 48 and 96 hours, the survival rates of both Paju-TLN and Paju-neo cells significantly differed from those at 24 hours(Paju-TLN: 48.69±5.33% (48 hours),27.91±4.24% (96 hours); and Paju-neo: 39.73±6.17%(48 hours), 19.53±5.15% (96 hours),P＜0.05)(Figure 2).

Figure 1 Telencephalin (TLN) reduces cell apoptosis in cultured Paju-TLN cells.Paju-TLN and Paju-neo cells were treated for 96 hours with 1 nmol/L of 42 amino acid long beta-amyloid peptide prior to fixation and analysis for Hoechst 33258 staining.The apoptotic phenotype was assessed by nuclear Hoechst 33258 staining(fluorescence microscopy) and morphological changes(phase contrast microscopy).The images are representative of four to five separate experiments.Scale bars: 20 μm.

Cell nuclei staining with Hoechst 33258

Following maintenance in normal culture conditions, a majority of nuclei showed blue fluorescence.Neither cell line showed signs of apoptosis 24 hours following normal culturing.Nevertheless, after Aβ treatment for 24 hours,some Paju-neo cell nuclei had pyknotic and blue round particles, indicating that apoptosis occurred (23.48 ±3.13%).Simultaneously, significantly fewer Paju-TLN cells presented with pyknotic or fragmented nuclei when compared to Paju-neo cells (11.56±2.79%,P＜0.05).Pyknosis increased over time, as did nuclear fragmentation and cell death.Paju-TLN cells displayed reduced apoptosis relative to Paju-neo cells at 48 and 96 hours (Paju-TLN: 24.23±3.43, 47.92±4.57; and Paju-neo: 36.77±4.08, 68.83±5.26, respectively,P＜0.01) (Figure 3).Following 96 hours of Aβ treatment, all Paju-neo cells died.

Figure 2 Telencephalin(TLN) promotes cell viability in cultured Paju-TLN cells.Paju-TLN and Paju-neo cells were treated with 1 nmol/L of the 42 amino acid long β amyloid peptide (Aβ42) for 24, 48, and 96 hours prior to performing the MTT assay.Aβ42 reduced cell viability in cultured Paju-neo cells.aP＜0.05, vs. Paju-TLN group.Values are expressed as mean±SD (n=6).The two-sample t-test was used.

Figure 3 Telencephalin(TLN) decreases the percentage of Paju-TLN apoptotic nuclei.Paju-TLN and Paju-neo cells were treated with 1 nmol/L of the 42 amino acid long beta-amyloid peptide (Aβ42) for 24, 48, and 96 hours prior to the addition of Hoechst 33258.Aβ42 reduced cell viability in cultured Paju-neo cells.aP＜0.05, vs.Paju-neo group. Values are expressed as mean±SD (n=6).The two-sample t-test was used.

Paju cell apoptosis

Figure 4 Apoptotic changes in Paju-telencephalin(TLN)and Paju-neo cells monitored by flow cytometry.The figures show that apoptotic rate of Paju-neo cells was 25.33% (A), and the rate for Paju-TLN cells was 19.15%(B) at 48 hours.TLN significantly reduced the apoptotic rate of Paju-TLN cells.Blue represents the apoptotic peak.

Paju-TLN and Paju-neo cells were treated for 48 hours with 1 nmol/L Aβ42prior to fixation and analysis by flow cytometry.Paju-TLN and Paju-neo cells exhibited similar apoptosis levels 24 and 48 hours following Aβ treatment.The apoptotic rates of Paju-TLN cells were 2.50% and 19.15%, and the rates for Paju-neo cells were 9.89% and 25.33% at 24 and 48 hours, respectively.The apoptotic rate of Paju-TLN cells was significantly less than Paju-neo cells (n=3,P＜0.05; Figure 4).

DISCUSSION

Paju cells derived from a human primitive neuroectodermal tumor have been extensively used in neurobiological studies[9-10].Aβ-treated cell cultures simulate AD and cause apoptosisviaactivation of death-stimulating factors, inflammatory factors, and oxidative stress.

TLN is an adhesion molecule expressed in cell bodies and dendrites of mammalian neurons around birth, with a more complicated structure than other ICAMs.TLN plays important roles in regulating immunological activity in the brain, synaptic development, and signal transduction[7,10-15].The carboxyl terminus of presenilin 1 and 2 (PS1 and PS2) binds to TLN.Remarkably, the amyloid precursor protein (APP) binds to the same regionsviapart of its transmembrane domain encompassing the critical residues mutated in familial AD[16].TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons[17].To date, no study has investigated the effects of TLN on Aβ-induced apoptosis.In this study, Paju-TLN cells were used to test the neuroprotective capacity of TLN in Aβ42-induced apoptosis.Paju-TLN cells remained viable for longer and few cells underwent apoptosis (P＜0.05).Morphological changes were delayed and Paju-TLN cells survived longer than Paju-neo cells during treatment.These results indicated that the expression of TLN protein prolonged cell tolerance following Aβ42treatment, even 48 and 96 hours after treatment.Results from nuclear staining and flow cytometry also suggested that the expression of TLN could delay or reduce Paju cell apoptosis induced by Aβ42.A previous study has linked a reduction in TLN to changes in dendritic spine morphology that are associated with long-term potentiation[18].Other related studies are listed in Table 1.

Table 1 Related studies

These studies have indicated that there is a close relationship between TLN and AD.Moreover, TLN may reduce Paju cell apoptosis induced by Aβ42and have a neuroprotective effect.

MATERIALS AND METHODS

Design

A cytologicalin vitrostudy.

Time and setting

Experiments were performed at the Central Laboratory,Xiangya Second Hospital, Central South University,China, from December 2008 to June 2009.

Materials

Cell lines

A human TLN gene transfected Paju-TLN cell line and Paju cell line were kindly supplied by the Life Science College, Helsinki University, Finland[10,22-23].A Paju-neo cell line was established in the laboratory with an empty pcDNA3.1 vector in the Gene Center of Second Xiangya Hospital, Central South University,China.

Methods

Paju cell culturePaju-TLN and Paju-neo cells were incubated in high-glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (Gibco, New York, NY, USA) supplemented with 10% (v/v) fetal bovine serum, 50 mg/L G418, and 2 mmol/L glutamine (Gibco).The medium was replaced every 3–5 days.Cells in the logarithmic phase were harvested for further use.

Peptide preparation and peptide treatment

The Aβ42peptide corresponding to the human Aβ wild type sequence (rPeptide, Japan), which was reconstituted according to the manufacturers’instructions, and treated with 0.1% (w/v) NH4OH/ PBS, at a stock concentration of 1 mmol/L, at 37°C for 7 days[24].The cells were grown to confluency at 37°C for 3 days and differentiated in Dulbecco’s modified Eagle’s medium containing phenol red.Cells were grown in 6-well, 24-well and 96-well plates at a density of 2 × 106,2 × 104and 2 × 104cells/cm2, respectively, in a humidified atmosphere containing 5% CO2(Hera Cell,Heraus, Germany).Cultured cells were treated with Aβ42(1 nmol/L) for different periods of time, ranging from 24 to 96 hours, as indicated in the figure captions[24].The peptides were added into culture medium on the 4thculturing day.The original supernatant solution was removed from the cells with a pipette and the new medium (0.3-3 mL, 37°C, containing aggregated Aβ42alone or without the peptide) was added rapidly (within 3 seconds) to the 6-well, 24-well and 96-well plates at 37°C.Aβ42was further diluted in culture medium(1 nmol/L) just before treatment.Sedimented Aβ clusters remained cell bound after cells were washed.

Observation of cell morphology

Following Aβ treatment, Paju-TLN and Paju-neo cells were observed under an inverted phase contrast microscope (Nikon, Tokyo, Japan) from 24 to 96 hours.

Paju cell viability as detected by MTT

Paju-TLN and Paju-neo cells were seeded at 1 × 104cells/mL in a 96-well plate, incubated in Aβ medium for 24, 48 and 96 hours, followed by incubation with MTT(Sigma, St.Louis, MO, USA) for 4 hours.After removal of the medium, the cells were dissolved in dimethyl sulfoxide (Sigma).Absorbance values were measured using a microplate reader at 490 nm and a reference wavelength of 630 nm.Blank wells without cells served as zero adjustment.

Apoptotic rate determined using Hoechst 33258 staining

Paju-TLN and Paju-neo cells were seeded at 104cells/mL in 24-well plates, and harvested after 24, 48,and 96 hours of Aβ treatment.Cells were washed once in PBS, fixed in 4% (w/v) paraformaldehyde for 20 minutes, rinsed twice in PBS, stained with 1 mg/mL Hoechst 33258 (Sigma) for 15 minutes in the dark,mounted, observed under a fluorescence microscope(Nikon, Tokyo, Japan), and photographed.Nuclei were counted (n=500), and the percentage of apoptotic nuclei was calculated[16-17].The percentage of apoptotic nuclei=(number of apoptotic nuclei/500) × 100%.

Apoptosis as determined by flow cytometry

Paju-TLN and Paju-neo cells were seeded at 1 × 106cells/mL in a 25 cm2incubator, harvested 24 and 48 hours following Aβ treatment, digested by trypsin(Sigma), rinsed twice in PBS, fixed in 75% (v/v) cold ethanol, and stored at 4 °C.Cells were initially stained with 100 μg/mL propidium iodide (Sigma) for 10 minutes and then analyzed by flow cytometry (BD Guava Grace Bio-Labs, USA).A total of 10 000 cells were detected and the apoptotic rate was calculated.

Statistical analysis

The data were analyzed using Microsoft Office Excel 2003 (No.11.8117.8122., USA) SP2 and SPSS 9.0 software (SPSS, Chicago, IL, USA).Results were expressed as mean±SD.Statistical analysis was performed using two-samplet-test (α=0.05).

Author contributions:Heping Yang provided data,participated in integration, study design and wrote the article.Dapeng Wu participated in data analysis.Xiaojie Zhang provided technical information.Xiang Wang participated in statistical processing.Yi Peng provided technical support.Zhiping Hu provided technical support and aided with manuscript preparation.

Conflicts of interest:None declared.

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