Neuroprotection against Amyloid- β-Induced DNA Double-Strand Breaks Is Mediated by Multiple Retinoic Acid-Dependent Pathways

Abstract

In this study, we have investigated the role of all-trans-retinoic acid (RA) as a neuroprotective agent against Aβ _1-42-induced DNA double-strand breaks (DSBs) in neuronal SH-SY5Y and astrocytic DI TNC₁ cell lines and in murine brain tissues, by single-cell gel electrophoresis. We showed that RA does not only repair Aβ _1-42-induced DSBs, as already known, but also prevents their occurrence. This effect is independent of that of other antioxidants studied, such as vitamin C, and appears to be mediated, at least in part, by changes in expression, not of the RARα, but of the PPARβ/δ and of antiamyloidogenic proteins, such as ADAM10, implying a decreased production of endogenous Aβ. Whereas Aβ _1-42 needs transcription and translation for DSB production, RA protects against Aβ _1-42-induced DSBs at the posttranslational level through both the RARα/β/γ and PPARβ/δ receptors as demonstrated by using specific antagonists. Furthermore, it could be shown by a proximity ligation assay that the PPARβ/δ-RXR interactions, not the RARα/β/γ-RXR interactions, increased in the cells when a 10 min RA treatment was followed by a 20 min Aβ _1-42 treatment. Thus, the PPARβ/δ receptor, known for its antiapoptotic function, might for these short-time treatments play a role in neuroprotection via PPARβ/δ-RXR heterodimerization and possibly expression of antiamyloidogenic genes. Overall, this study shows that RA can not only repair Aβ _1-42-induced DSBs but also prevent them via the RARα/β/γ and PPARβ/δ receptors. It suggests that the RA-dependent pathways belong to an anti-DSB Adaptative Gene Expression (DSB-AGE) system that can be targeted by prevention strategies to preserve memory in Alzheimer's disease and aging.

Figure 1

All-trans-retinoic acid (RA) protects against Aβ-induced DSBs in SH-SY5Y and astrocytic DI TNC₁ cells as well as in the neocortex of young and aged male C57BL/6J mice. Representative pictures of comets with various tail lengths of (a) SH-SY5Y cells, of (b) DI TNC₁ cells, and of cortical tissues originating from (c) young (4 months; n = 3 mice) or (d) aged (16 months; n = 3) mice following RA (5 μM) and/or Aβ (20 μM) in vitro treatments for 2 × 30 min (SH-SY5Y cells; cortical tissue) and 2 × 1 h (DI TNC₁ cells). ø = without treatment; scale bar: 200 μM. Box plots of mean comet tail lengths of (e) SH-SY5Y cells (number of cells measured: 33 < n < 52), of (f) DI TNC₁ cells (55 < n < 72), of (g) 3 young (30 < n < 55), and of (h) 3 aged mice (31 < n < 57). ANOVA with Bonferroni correction: one experiment for SH-SY5Y cells (replicated in Figure 4(e)) and 3 for DI TNC₁ cells; ^∗p < 0.05 for all experiments or all three mice; ⁺p < 0.05 for 2 out of 3 mice; ^◊p < 0.05 for 1 out of 3 mice.

Figure 2

Effects of antioxidants against Aβ-induced (20 μM) DSBs in SH-SY5Y cells compared to all-trans-retinoic acid (RA; 5 μM) treatment. In (a), example of comet assay with 1 h treatments with Aβ, RA, and/or glutathione (glut). Scale bar: 200 μM. In (b) to (e), data analyses: (b) glutathione (1 μM), (c) α-tocopherol (toco; 1 μM), (d) L-carnosine (carn; 100 μM), and (e) vitamin C (vitC; 100 μM). Box plots of mean comet tail lengths (number of cells measured: 18 < n < 53). ANOVA with Bonferroni correction: the experiments with L-carnosine and vitamin C were repeated 3 times (^◊p < 0.05 for 1 experiment out of 3; ⁺p < 0.05 for 2 experiments out of 3; ^∗p < 0.05 for 3 experiments out of 3) whereas the experiments with glutathione and α-tocopherol were carried out 1 time (^∗p < 0.05). ø = without treatment.

Figure 3

Changes in protein expression following 1 μM all-trans-retinoic acid (RA) treatment of the deep and superficial neocortical layers, as well as of the hippocampus, of (a) three 1-month-old and (b) three 17-month-old male C57BL/6J mice. The proteins are involved in the amyloid cascade (Presenilin 1 or PS1/γ-secretase, BACE1/β-secretase, ADAM10/α-secretase, and APP C-terminus), in Tau phosphorylation (phospho-Tau (AT8) or unphosphorylated Tau (Tau1)), in synaptic functions (PSD95, GluN2B/NR2B), or in RA-dependent pathways (RARα, PPARβ/δ). GAPDH and DB71 stainings were used to demonstrate equal loading of the Western blot gels. Overall, we observed significant increases (see Section 3.3) of ADAM10, APP, and phosphorylated and unphosphorylated Tau proteins, suggesting the activation of neuroprotective mechanisms following the RA treatment, whereas the expression of the enzymes of the amyloidogenic pathway, PS1 and BACE1, or, in most cases, of the RA receptors was not increased. Protein sizes are indicated on the right. A size range is given for Tau isoforms (AT8 and Tau1).

Figure 4

(a) Schematic representation of the RA and 9-cis RA receptors, RAR, PPAR, and RXR, and their antagonists, AGN 193109 (AGN) for RARα,β,γ and GSK 3787 (GSK) for PPARβ/δ, as well as their neuroprotective effect on Aβ-induced double-strand breaks (DSBs). (b) Effects of 5 μM all-trans-retinoic acid (RA) against Aβ-induced DSBs and of 20 μM Aβ on DSB production in the presence of the transcription inhibitor actinomycin D (Acti; 1 μg/mL) and of the translation inhibitor cycloheximide (CHX; 10 μg/mL) in SH-SY5Y cells after a 1 h treatment. The inhibitors do not interfere significantly with the effect of RA whereas they do with Aβ. Box plots of mean comet tail lengths of SH-SY5Y cells (number of cells measured: 27 < n < 50). ANOVA with Bonferroni correction: the experiment was repeated 3 times (^◊p < 0.05 for 1 experiment out of 3; ⁺p < 0.05 for 2 experiments out of 3; ^∗p < 0.05 for 3 experiments out of 3). (c) Box plots of mean comet tail lengths of SH-SY5Y cells (31 < n < 53) following a 1 h treatment with RA, 9-cis RA (5 μM), and/or Aβ in the presence of AGN (50 μM) or not. (d) Box plots of mean comet tail lengths of SH-SY5Y cells (36 < n < 50) following a 30 min treatment with Aβ, RA, AGN, and GSK (10⁻⁵ M) and a combination of these factors. (e) Box plots of mean comet tail lengths of SH-SY5Y cells (31 < n < 34) following a 30 min treatment with RA, AGN, and/or GSK, and after a washing step, a second 30 min treatment with Aβ or not. (c–e) ANOVA with Bonferroni correction: ^∗p < 0.05. ø = without treatment.

Figure 5

Neuroprotection experiments carried out with SH-SY5Y cells to show the activation of RA-dependent pathways by a 10 min RA treatment or not (ø), followed by a 20 min treatment with Aβ or not (ø10′–ø20′, ø10′–Aβ20′, RA10′–ø20′, and RA10′–Aβ20′). (a) By immunofluorescent cytochemistry, it was shown that PPARβ/δ (green) increased in the cytoplasm with the RA–Aβ treatment, whereas a cytoplasmic increase of the RXRα/β/γ (red) could not be observed. However, sites of colocalisation (white arrows, green-yellow signals) could be detected (Merge, PPAR-RXR, or all channels). Signals were too faint in the cell nucleus (DAPI). (b) Duolink™ proximity ligation assay was used to determine PPARβ/δ and RXRα/β/γ heterodimerization under the same conditions as in (a). A significant increase of PPAR-RXR signals was observed with the RA–Aβ treatment only in the cytoplasm when compared to the ø–ø (p = 0.0125) or ø–Aβ (p = 0.0145) treatments. Only a nonsignificant (ns) increase was observed in the cell nuclei. The number of pictures analysed is 6 to 9. Similar experiments for RARα and RXRα/β/γ resulted in no significant differences. Scale bar for (a): 25 μM and for (b): 13 μM.