Purported Interactions of Amyloid-β and Glucocorticoids in Cytotoxicity and Genotoxicity: Implications in Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is a complex neurodegenerative disorder characterized by the presence of aggregates of the amyloid-β peptide (Aβ) that are believed to be neurotoxic. One of the purposed damaging mechanisms of Aβ is oxidative insult, which eventually could damage the cellular genome. Stress and associated increases in glucocorticoids (GCs) have been described as a risk factor for the development of AD, although the purported genotoxic effects of GCs have not been fully characterized. Therefore, it is possible to speculate about purported synergistic effects of GCs on the Aβ-driven genotoxic damage. This in vitro study addresses the single and combined cyto/genotoxic effects of Aβ and GCs in SH-SY5Y cells. Cytotoxicity was determined by the MTT assay, and the genotoxic effects were studied using the comet assay. A comet assay derivation allows for measuring the presence of the FPG-sensitive sites (mainly 8-oxoguanines) in the DNA, apart from the DNA strand breaks. Treatment with Aβ (10 μM, 72 h) induced cytotoxicity (35% decrease in cell viability) and DNA strand breaks, but had no significant effect on oxidative DNA damage (FPG sites). Corticosterone showed no effect on cell viability, genotoxicity, or reparation processes. Corticosterone was unable to neither reverse nor potentiate Aβ driven effects. The present results suggest the existence of alternative mechanisms for the Aβ driven damage, not involving oxidative damage of DNA. In addition, could be suggested that the interaction between Aβ and GCs in AD does not seem to involve DNA damage.

Keywords

Comet assay DNA breaks DNA damage DNA-formamidopyrimidine glycosylase oxidative stress

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disorder that results in progressive cognitive deficits. Aging is the primary risk factor for AD. Neuropathologically, AD brain is characterized by the accumulation of neurofibrillary tangles and senile plaques [1, 2]. Plaques are mainly composed by amyloid-β peptide (Aβ) which has shown to be deleterious for neurons [3]. In fact, the main current therapeutic strategies for AD target Aβ accumulations, either by decreasing its production [4] or by increasing its degradation [5].

Even though it has been suggested that Aβ induces DNA damage [6, 7], the mechanisms responsible for Aβ-driven genotoxic damage are not fully understood [8]. One of the proposed mechanisms to explain Aβ toxicity is mitochondrial dysfunction and the resulting oxidative damage [9, 10], which could eventually damage cellular genome. It has been described that AD brains showed an increase in oxidative species compared to control subjects [11], which in turn could be the cause of greater levels of DNA damage in postmortem brain analysis [12]. Moreover, DNA damage in peripheral blood lymphocytes is also increased [13, 14], even in preclinical AD stages [15].

Among the well-known risk factors contributing to the development of AD are chronic stress and the resulting increase in glucocorticoids (GC) [16 –19]. AD patients show a significant increase in GC levels [20], which could be worsening the AD the progression of the illness. Moreover, it has been described in transgenic mouse models of AD that GC exacerbated AD-like neuropathology [21, 22]. GCs have been shown to potentiate hippocampal damage induced by various noxious insults [23] including Aβ [24]. Interestingly, GCs induce mitochondrial dysfunction and oxidative damage in the brain [25]. Flint and colleagues [26] showed that GCs may also trigger genotoxic effects, although these effects are controversial [27].

Altogether, it is possible to speculate about purported synergistic effects of GCs on the Aβ driven genotoxic damage. This study addresses the single and combined genotoxic effects of Aβ and GCs in cell culture. The present results preclude oxidative damage as causal agent of the genotoxic effects of Aβ. In addition, GCs do not seem to interact with Aβ to induce DNA damage.

MATERIALS AND METHODS

Cell culture

Human SH-SY5Y neuroblastoma cells were cultured at 37°C, 5% CO₂, in Dubbelco‘s Minimum Essential Medium (DMEM; Gibco, ref.41966) supplemented with 10% fetal bovine serum (FBS; Gibco, 10500), and Penicillin/Streptomycin solution (Lonza, 17-602E) at 100 U/mL. For all experiments 70–80% confluent cultures were used. In these cells, the presence of GC receptors has been demonstrated by western blotting (Supplementary Figure 1).

Treatments

Aβ peptide fragment 25–35 (Aβ) (Sigma, A4559) was dissolved in sterile water to a stock concentration of 1 mM. Aβ was diluted in DMEM without phenol red (Gibco, 31053), to its final concentration and kept at 37°C for 4 days. After that time, the medium was supplemented with GlutaMax^® (Gibco, 35050), pyruvate (Gibco, 11360) and 1% FBS. SH-SY5Y cells were exposed to Aβ for short (24 h) or long (72 h) treatments. Corticosterone was daily fresh prepared (Sigma, C2505) in DMEM with 1% dimethylsulfoxide (DMSO) and 1% FBS. Exposure of cell to corticosterone (single or combined treatments) was 24 h. In the combined treatments, corticosterone was added simultaneously to Aβ (24 h treatment) or in the last 24 h of the Aβ treatment (72 h).

Cell proliferation assay

After finishing treatments, cells were rinsed with PBS and left growing undisturbed in fresh 10% FBS supplemented DMEM for 4 days. Cells were counted using trypan blue-based specific chambers (Life technologies, C10228) for Countess^® automated cell counter (Life technologies). For each treatment, the average growth rate was obtained and viability was calculated as the percentage relative to the average growth rate of controls.

MTT assay

MTT assay was performed as previously reported [28, 29]. A 5 mg/mL stock solution of MTT was prepared in PBS. After finishing the treatment, cells were rinsed with DPBS (Gibco, 14190), and 0.5 mg/mL MTT medium solution was added (Sigma, M5655). After 2 h incubation, medium was removed and the precipitated formazan was dissolved in pure DMSO. Spectrophotometric reading was conducted at 540 nm (Spectra MR, Dynex technologies). Survival was calculated as a percentage relative to the control sample.

Comet assay: DNA breaks and oxidative damage assays

Cells were embed in an 1% agarose (Sigma, A9414) matrix, placed on 1% agarose (normal melting point) pre-coated slides and left overnight in a lysis solution (2.5 M NaCl, Panreac, 131859; 0.1 M Na2-EDTA, Sigma, E5134; 10 mM Trizma base, Sigma, T1503; 1% Triton X-100, Sigma, T8787; pH 10). Samples were then treated with either formamidopyrimidine DNA glycosylase FPG in an enzyme reaction buffer (buffer F 0.1 M KCl 0.5 mM Na₂EDTA, 40 mM HEPES and 0.2 mg/mL bovine serum albumin, pH 8.0) or buffer solely [30], for 30 min at 37°C. Electrophoresis was performed at 1.5 V/cm for 20 min, in electrophoresis buffer (0.3 M NaOH, 1 mM Na2-EDTA, pH > 13). Slides were dyed with 4^′,6-diamidino-2-phenylindole DAPI, and comet tails were quantified using a semi-automated software (Comet Assay v.4.2, Perceptive Instruments). A positive control was introduced in all experiments (SH-SY5Y cells treated with 1 μM of the photosensitiser Ro 19-8022 + 5 min light exposition). In all cases, the positive control gave rise to positive results (data not shown). The median of the tail intensity percentage of 100 comets was taken as the representative value in each slide. For DNA breaks, we consider the median value of the slides treated without FPG. The oxidative damage score (i.e., FPG sensitive sites) was taken as the difference between slides treated with and without FPG. Due to the high number of samples, for the experiments confronting Aβ and corticosterone treatments, a minigel derivation was performed (6 samples per slide instead of only 1). This derivation requires a final normalization step, performed as described previously [31].

When checking the inhibition of reparation by GCs, cells were treated with corticosterone for 24 h, and after embedding them into agarose, cells were presented to a well-established DNA-strand break inducer [32], such a hydrogen peroxide (H₂O₂) solution (100 mM in DPBS) for 1 min. After rinsing in fresh DPBS, cells were placed in fresh medium DMEM solution (supplemented with 10% FBS) for 0, 10, 30, or 60 min. After that, cell lysis and the continuing steps were performed as described before.

Statistical analysis

In all cases the number of cases is the mean of a technical quadruplicate. Statistical analysis was performed in R software (version 3.2.2) implemented in R Studio (version 0.99.486). Data was analyzed by one-way or two-way ANOVA followed by Bonferroni’s test when necessary. The significance level was set at p < 0.05.

RESULTS

Cytotoxic and genotoxic effects of Aβ

Aβ (0.1–10 μM) revealed cytotoxic effects in the MTT assay only in the 72 h treatment (Fig. 1B) [One-way ANOVA: F_(3,10) = 1.30, p = 0.33 (24 h), F_(3,11) = 4.52, p < 0.05 (72 h), *Bonferroni’s post hoc test, control versus Aβ 10 μM, p < 0.05, n = 4 in each concentration]. This effect seems to be related to cytotoxic effects, as Aβ showed no effect (Fig. 1D) in the cell proliferation assay [One-way ANOVA: F_(3,11) = 1.284, p = 0.33 (24 h), F_(3,12) = 0.55, p = 0.66, (72 h) n = 4].

24 h of exposition to Aβ did not induce either strand breaks (Fig. 1E) [One-way ANOVA, F_(3,21) = 0.78, p = 0.52, n = 7] or oxidative damage (Fig. 1G) [One-way ANOVA, F _(3,22) = 0.51, p = 0.68, n = 7]. In contrast, 72 h treatment induced strand breaks production (Fig. 1F) [One-way ANOVA, F _(3,21) = 3.839, p = 0.02, n = 7 *Bonferroni’s post hoc test, control versus Aβ 10 μM, p < 0.05] with no effect in oxidative damage (Fig. 1H) [One-way ANOVA, F _(3,12) = 1.384, p = 0.2950, n = 4].

Lack of effect of corticosterone treatment on cell viability, genotoxicity, or reparation processes

The effects of corticosterone were checked (24 h) at concentrations ranging from 0.01 μM to 10 μM. Corticosterone treatment did not show cytotoxic effects either in the MTT assay (Fig. 2A) [One-way ANOVA; F_(4,15) = 2.62; p = 0.0765, n = 4] nor in the proliferation assay (Fig. 2B) [One-way ANOVA, F_(4,9) = 0.19, p = 0.93; n = 3). Very short time effects of corticosterone (3 h) were shown ineffective as well (Supplementary Figure 2).

In the comet assay, corticosterone was ineffective in producing DNA strand breaks [Fig. 2C, F_(4,10) = 0.057; p = 0.99, n = 3), or altering FPG sensitive sites (Fig. 2D) [F _(4,10) = 0.22; p = 0.92, n = 3).

Lastly, it was checked a purported indirect genotoxic effect of corticosterone through altering the mechanisms of reparation of the damage induced by a genotoxic agent, such as H₂O₂. The exposure to corticosterone did not alter the repairing curve to H₂O₂-induced damage (Fig. 3A) [One-way ANOVA, repeated measures, interaction effect, F _(12,30) = 0.62, p = 0.81; n = 3). As an internal control, slides treated similarly but lacking hydrogen peroxide incubation step did not show any repairing trend (Fig. 3B) ([One-way ANOVA, repeated measures, interaction effect, F _(12,30) = 0.47, p = 0.91, n = 3].

Effects of a combined treatment of Aβ with corticosterone

Cell proliferation was not affected by a combined treatment of Aβ (1 and 10 μM) with corticosterone (0.01 and 1 mM) in a short treatment time (24 h) (Fig. 4A) [Two-way ANOVA, F _(6,33) = 1.17 p = 0.35, n = 3]. After longer treatment (72 h), Aβ increased the proliferation at 1 μM concentration (Fig. 4B) [Two-way ANOVA, F _(6,24) = 0.59, p = 0.74, *Bonferroni’s post hoc test, control versus Aβ, 1 μM, p < 0.05, n = 3].

Firstly we checked a purported genotoxic effect of corticosterone at non deleterious time/concentrations of Aβ (1 and 10 μM, 24 h). The combined treatment did not induce any effects in DNA strand breaks (Fig. 4C, two-way ANOVA, F _(6,63) = 0.09, p = 0.99, n = 7) or oxidative damage (Fig. 4E, two-way ANOVA, F _(6,63) = 0.15, p = 0.99, n = 7).

We then evaluated the interacting effects 72 h of exposition to Aβ with corticosterone. Even though a combined treatment of Aβ+corticosterone increased the DNA strand breaks in the comet assay (Fig. 4D), this effect was associated with a main effect of Aβ [Two-way ANOVA, main effect of Aβ, F _(3,64) = 4.76, p < 0.01, n = 6, Bonferroni’s post hoc test, control versus Aβ, 10 μM, p = 0.01), and corticosterone was unable to either reverse nor potentiate Aβ driven effects. FPG sensitive sites were unaltered by any treatment (Fig. 4F) [Two-way ANOVA, F_(6,36) = 0.36, p = 0.89, n = 6].

DISCUSSION

This study aimed to characterize the cytotoxic and genotoxic effects of Aβ. As a second objective, it is studied here the hypothesis that GCs could further potentiate Aβ-driven deleterious effects.

The comet assay is a reliable method to evaluate DNA damage in a single-cell resolution performance [33, 34]. It has shown to be useful in in vitro and in vivo experiments and has been successfully used in a wide range of scientific fields like toxicology, neuroscience, or ecology. Alterations in DNA bases can be detected by digesting agarose embed cell lysates. Just after lysis, enzymes (for instance, FPG) induce basic sites in specific lesions that will lead to breaks by the procedure [35]. Alkylated bases have been studied in this way [36], as well as UV irradiation damage like cyclobutane pyrimidine dimers [37]. FPG, a prokariotic DNA repair enzyme, removes 8-oxoguanines and other purine oxidation products. This allows us to use FPG as a tool to evaluate oxidative damage, as 8-oxoguanine is one of the main products of DNA oxidation [38]. DNA fragmentation occurs during apoptosis and cell death [39]. It is possible that the earliest stages of apoptosis or necrosis might give rise to increases in DNA in comet tail (although subsequent fragmentation is usually too severe to be detected). It is advisable to ensure that the experimental conditions are not such as to induce cell death through mechanisms other than genotoxicity, to avoid false positive evaluation in the comet assay. Therefore, cytotoxic concentrations (in means of viability) of damaging agent should be avoided [31]. In our study, dosages and exposition times were carefully chosen to avoid these conflicting effects.

There are strong limitations in using in vitro models to address highly complex in vivo issues. This aspect indicates that the results here shown should be evaluated carefully, and their potential implications in actual physiopathology of Alzheimer’s disease must be taken carefully. Nonetheless, in vitro cell culture assays possess potential strengths, especially concerning two research challenges. First, new insights into the mechanisms of action of the effects of new and old compounds can be proposed, as they are much more straightforward assays. Second, they can be used to evaluate potential toxic activities, where in vitro assays might be considered equivalents to in vivo assays [40, 41]. Although in vitro results will always be surrogated to in vivo confirming experiments, they are powerful tools to address these two topics, all of which have been evaluated in the present article (i.e., toxicity and mechanisms).

Aβ is a toxic peptide that has been shown to trigger oxidative damage, both full-length species, like Aβ_1 - 42 [7], or the fragment Aβ_25 - 35 [6 , 42]. Despite not being a naturally occurring peptide, the peptidic fragment corresponds to the amino acid 25 to 35 of Aβ, which exhibits large β-sheet aggregated structures comparable to oligomeric Aβ and retains the toxicity of full length peptide [43 –45], and it is consider the functional toxic domain of the full-length peptide [46]. In our hands, an exposition time of 72 h was needed to obtain cytotoxic effects, even though previously published works showed that 24 h of Aβ exposition was able to cause significant cell death in a SH-SY5Y cell line [6, 42]. At this point, it is important to note the importance of the Aβ preparation protocol, as subtle differences in the preparation methods would yield different proportions of aggregated Aβ species [47]. It has been shown that early formed pre-fibrillar aggregates of Aβ are mainly endowed with cytotoxicity, whereas mature fibrils are much less toxic or even harmless [48].

Aβ genotoxic effects have been attributed to its capability to increase oxidative damage. Aβ_25 - 35 has been shown to increase intracellular reactive oxygen species [49] and, as a response mechanism, antioxidant enzymes [42]. There is even some evidence of oxidative damage preceding Aβ accumulation in the brain [50, 51]. However, the therapeutic benefits of addressing oxidative stress (with antioxidants) have not been successfully achieved yet in AD [52]. It has been previously shown that Aβ_25 - 35 exposure (10 μM) increases DNA damage and decreases cell viability in both PC12 cells [42] and SH-SY5Y cells [49]. Although there was a trend toward increases in the FPG sensitive sites induced by Aβ after 24 and 72 h, they were non-significant. In contrast, clear increases in DNA breaks were found after exposure to Aβ after 72 h. Supporting our results, Forestier et al. [53], using a model of overproduction of Aβ by overexpressing APP751 (carrying APPswe mutation) in an SH-SY5Y cell line, demonstrated an increase in strand breaks. Therefore, it is suggested the existence of mechanisms, other than oxidative damage, as being associated wtih the genotoxic effects of Aβ. It has been demonstrated that Aβ is able to interact directly with DNA [54]. It is also possible to speculate that oxidative damage either is not able to damage DNA integrity, or the damage is rapidly repaired with no deleterious consequences. It has been shown that APP overexpression increased the genotoxic effects caused by different oxidant agents, such as H₂O₂ or sulfate cupper, suggestive of the presence of reduced expression of repairing enzymes involved in 8-oxoguanine reparation [53]. Therefore, although Aβ might not be inducing DNA oxidation per se, it could be indirectly increasing this damage in experimental conditions in which other oxidant agents are present.

Concerning GCs, conflicting results have been described in literature. Supporting the present results on the lack of effect of corticosterone treatment to affect cell viability or genotoxicity, Jorgensen and colleagues [27] found no positive effects of DNA damage induced by GCs in vivo. However, other reports have found that GCs were able to induce strand breaks in mouse fibroblasts [26]. In addition to methodological questions, it is of note that in the different cell line used in that work (T3T), the presence of GC receptors has not been demonstrated to our knowledge, whereas in SH-SY5Y, we verified the GR expression. It has also been postulated that GCs could inhibit repairing mechanisms, leading to DNA damage [26], but the amelioration by GCs of oxidative DNA damage induced by different toxics has also been suggested [55]. In our hands, no effect (positive or negative) of corticosterone on the reparation processes was found.

Finally, we examined the hypothesis that even though GCs have no genotoxic effect per se, they could interfere with Aβ deleterious effects. Estrogens, which are structurally related to GCs, are protected from the genotoxic effects of Aβ by recruiting the protective agent Hsp70 to the nucleus [6]. Hsp70 is a known protector of DNA integrity [56], and it has also shown to interact with GC receptors [57]. However, in our hands, corticosterone showed no effect to either potentiate (24 h experiments) or inhibit (72 h experiments) the genotoxic effects of Aβ.

CONCLUSIONS

The present work supports previously reported data on the cytotoxic effects of Aβ. However, the present results suggest the existence of alternative mechanisms for the Aβ-driven damage, which does not seem to involve oxidative damage of DNA. In addition, even though chronic stress and associated increases in GCs are widely accepted as risk factors contributing to the development or maintenance of AD, from our results it could be suggested that the interaction between Aβ and GCs does not involve DNA damage. Due to the many deleterious mechanisms of Aβ and the promiscuous effect of the GCs in cell biology, interactions between them could be expected, although involving different molecular or biochemical mechanisms.

Footnotes

ACKNOWLEDGMENTS

This work has been supported by a grant to MJR from ISCIII–Subdirección General de Evaluación y Fomento de la Investigación (FIS 13/00858), co-financed by the European Union (Fondo Europeo de Desarrollo Regional, FEDER) “Una manera de hacer Europa“ and to AA from Ministerio de Economía y Competitividad (‘Ramón y Cajal’ programme, 2013) of the Spanish Government. XB is fellow from the “Asociación de Amigos, University of Navarra”. The authors thank Dr. A. Collins for the generous gift of FPG and Roche for the positive control of Ro.

Authors’ disclosures available online ().

Appendix

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