Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

Abstract

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.

Keywords: Alzheimer disease; BCL2; BECN1; apoptosis; autophagy; soluble β-amyloid 42.

Figure 1. Treatment of differentiated SK-N-BE cells with 1 μM Aβ1-42 monomers and oligomers significantly increase BECN1 and LC3-I and LC3-II fluorescence intensity and protein levels. (A) Cells treated with 1 μM Aβ42 monomers and oligomers for 24 h were stained, after fixation and permeabilization, with BECN1 or LC3 antibodies. After washing in PBS, sections were incubated with secondary antibodies raised in different species: 1:200 cyanine 3-conjugated donkey anti-mouse secondary antibody and 1:100 cyanine 2-conjugated anti-rabbit secondary antibody with 1% BSA in PBS. For counterstaining, cells were incubated for 3 min with DAPI diluted 1:50 in methanol 0.1 M and rinsed with PBS. After 24 h both monomeric and oligomeric Aβ1-42 preparations increased BECN1 immunoreactivity; however a higher number of positive cells were observed after treatment with the monomeric preparation. (B) BECN1 and LC3-I/-II protein levels in cells treated with 1 μM Aβ42 monomers and oligomers at different time points up to 48 h. Both preparations were able to significantly increase BECN1 levels, but the increase was more pronounced with monomers than with oligomers (up to 3-fold with monomers vs. 2.5-fold with oligomers). Examination of LC3-II levels also showed an increase in protein levels for both monomeric and oligomeric preparations. Monomers of Aβ1-42 led to a marked and significant increase in LC3-II protein levels that started at 3 h and was still present after 48 h of treatment, whereas oligomers produced a transient increase that was no longer present after 8 h to 12 h of incubation. The error bars represent standard deviations. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 2. Treatment of differentiated SK-N-BE cells with 1 μM Aβ1-42 monomers, but not oligomers, significantly decreased autophagic flux, through accumulation of early endosomes. (A) SQSTM1 protein levels in cells treated with 1 μM Aβ1-42 monomers and oligomers up to 48 h. Treatment of cells with monomeric preparations was followed by a significant (> 2-fold) increase in SQSTM1 levels after 1 h of incubation that lasted up to 24 h, whereas oligomers of Aβ1-42 caused a slight, but not significant decrease in SQSTM1 protein levels. (B) On the left, confocal images of representative cultured SK-N-BE neuronal cells transfected with GFP-RFP-LC3 DNA for 24 h following 8 h of treatment with 1 μM Aβ1-42 monomers or oligomers. On the right, quantification of early and late autophagosomes per cells (15 cells) after treatment with 1 μM Aβ1-42 monomers and oligomers. The increase in number of early autophagosomes (GFP⁺-RFP⁺) was much higher in cells treated with monomeric preparation than in cells treated with oligomers, whereas late endosomes (GFP^- RFP⁺) were significantly increased after treatment with both preparation. For counterstaining, cells were incubated for 3 min with DAPI diluted 1:50 in methanol 0.1 M and rinsed with PBS. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 3. Treatment of differentiated SK-N-BE cells with 1 μM Aβ1-42 monomers affects autophagic flux through impairment of lysosomal activity. (A) Lysosomal-associated membrane protein 1 (LAMP1) immunostaining after 24 h treatment with the Aβ species. Cells treated with 1 μM Aβ42 monomers and oligomers for 24 h were stained, after fixation and permeabilization, with LAMP1 monoclonal and Aβ42 polyclonal antibodies. After washing in PBS, sections were incubated with secondary antibodies raised in different species: 1:200 cyanine 3-conjugated donkey anti-mouse secondary antibody and 1:100 cyanine 2-conjugated anti-rabbit secondary antibody) with 1% BSA in PBS. For counterstaining, cells were incubated for 3 min with DAPI diluted 1:50 in methanol 0.1 M and rinsed with PBS. As shown, lysosomal vesicles are formed and also there is a colocalization with lysosomes and the 2 Aβ1-42 preparations. (B) CTSD activity evaluation in SK-N-BE differentiated cells after treatments with the 2 Aβ1-42 preparations. Only treatment with Aβ1-42 monomers was followed by a significant decrease in the CTSD activity after 6 h and up to 24 h. Pepsatin, used as a positive control induced ~80% inhibition of the enzyme activity after 8 h of incubation. (C) LC3-II protein levels in SK-N-BE cells pretreated with 100 µM chloroquine for 24 h and then treated with Aβ1-42 oligomeric preparation for 8 h. Pretreatment of cells with the lysosomal inhibitor was able of mediating a strong and significant increase in LC3-II protein levels, with a trend almost similar to monomers. The error bars represent standard deviations. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 4. Treatment of differentiated SK-N-BE cells with 1 μM Aβ1-42 oligomers, but not monomers, significantly increases LDH release in the medium as well as cleavage of CASP3 and DNA fragmentation. (A) LDH release in differentiated SK-N-BE treated with Aβ1-42 oligomers as well as monomers for 24 h. The treatment of differentiated neuroblastoma cells with oligomeric Aβ1-42 preparation induced a more severe (~60% vs. 20%) release of LDH compared with the monomeric form. (B) Cleaved-CASP3 protein levels in cells treated with different Aβ species. We found that only oligomeric Aβ1-42, significantly increased (2.5-fold) the cleavage of CASP3 at 3 h and 6 h of incubation. (C) Cells treated with both oligomers and monomers of Aβ1-42 for 6 h were fixed, permeabilized, and stained with polyclonal antibodies against cleaved CASP3 (red) or with the DNA fragmentation stain TUNEL (green), and then analyzed by confocal microscopy. Monomers did not change cell number compared with controls, whereas oligomers strongly decreased it (oligomers: 61% increase in apoptotic nuclei vs. control; monomers: 18% increase in apoptotic nuclei vs. controls). Arrows indicate puncta of either cleaved CASP3 or TUNEL staining. Higher magnification views of given areas are encapsulated in squares. The nuclear stain DAPI (4,6 diamidino-2phenylindole, blue) was used to stain cells. Scale bars: 100 μm, or 15 μm for higher magnification views. The error bars represent standard deviations. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 5. Treatment of differentiated SK-N-BE cells with 1 μM Aβ1-42 oligomers significantly increases CYCS and BAK1 and decreases BCL2 levels and allows the interaction between BCL2 with BECN1. (A) CYCS, BAK1 and BCL2 protein levels in differentiated SK-N-BE cells treated with Aβ1-42 oligomers as well as monomers up to 24 h. Treatment with oligomeric Aβ1-42 increased the CYCS release (100 - 120%) after 3 h and up to 24 h, and BAK1 protein levels (120–150%) after 1 h and up to 24 h, whereas BCL2 levels resulted significantly decreased (50%). Treatment of cells with monomeric preparations of Aβ1-42 did not affect these parameters. (B) Cells treated with Aβ preparations were immunoprecipitated with polyclonal BECN1 antibody and revealed with monoclonal BCL2 antibody. We found that BCL2 is linked to BECN1 only after 3 h to 6 h of treatment with Aβ1-42 oligomers. (C) BCL2 and cleaved CASP3 protein levels in differentiated SK-N-BE control and overexpressing BCL2 cells treated with 1 μM Aβ1-42 oligomers. BCL2 overexpression was able to attenuate the cleavage of CASP3 mediated by oligomers. The error bars represent standard deviations. Experiments were conducted in triplicate. **, significantly different from controls (P < 0.02).

Figure 6. Treatment of differentiated SK-N-BE cells with 1 μM Aβ1-42 monomers increases BACE1 expression and activity through the activation of the MAPK/JNK pathway. (A) Nuclear pMAPK/JNK protein levels in differentiated SK-N-BE treated with Aβ1-42 monomers up to 24 h. The treatment was followed by a robust activation of the MAPK/JNK pathway, as shown by the significant increase (~3 to 2.5-fold) in levels of phospho- MAPK/JNK in nuclear fractions. (B) Phosphorylated BCL2 protein levels of in cytosolic fractions of cells treated with monomers up to 8 h. As expected with the activation of the MAPK/JNK pathway, cells treated with Aβ1-42 monomers revealed a significant increase (~2.5-fold) in BCL2 phosphorylation (C–E) BACE1 expression, protein levels, and activity in cells treated with Aβ1-42 monomeric preparation. Treatment of cells with Aβ1-42 monomers induced a 1.5- to 2.5-fold increase in BACE1 mRNA, as well as a 100% to 200% increase in BACE1 protein levels and a 35% increase in BACE1 activity. The error bars represent standard deviations. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 7. Treatment of differentiated SK-N-BE cells with 1 μM Aβ1-42 oligomers does not affect BACE1 expression and the JNK pathway. (A) Nuclear pJNK protein levels in differentiated SK-N-BE treated with Aβ1-42 oligomers up to 24 h. Aβ1-42 oligomers do not activate JNK phosphorylation in nuclear fraction of differentiated SK-N-BE cells. (B–D) BACE1 expression, protein levels, and activity in cells treated with Aβ1-42 oligomeric preparation. Treatment of cells with Aβ1-42 oligomers was not followed by significant induction of BACE1 mRNA, protein levels, and activity. The error bars represent standard deviations. Experiments were conducted in triplicate.

Figure 8. Treatment of differentiated SK-N-BE cells with different concentrations of Aβ1-42 monomers or oligomers does not modify the effects observed with the 1 µM concentratiob. BACE1 and CASP3 protein levels in differentiated SK-N-BE cells treated with a range (100 nM–5 µM) of Aβ1-42 monomers or oligomers for 6 h. We observed that monomers are not capable of increasing CASP3 levels even at higher concentrations, whereas they increase BACE1 protein levels at any concentration. Oligomers, in turn, fail to increase BACE1 levels at any concentration, but significantly increase cleaved CASP3 protein levels at all concentrations. The error bars represent standard deviations. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 9. BECN1 is needed for the toxicity mediated by Aβ1-42 oligomers. (A) BECN1, BCL2, and cleaved CASP3 protein levels in SK-N-BE cell lines stably expressing a specific shRNA against BECN1 or scrambled shRNA treated with Aβ1-42 oligomers up to 6 h. BECN1 is observed only in cells expressing scrambled shRNA while a band corresponding to the protein is not observed in cell expressing shRNA against BECN1. BCL2 protein levels remain unchanged in cells silenced for BECN1 treated with Aβ1-42 oligomers up to 6 h, whereas cleaved CASP3 are protected but slightly significant with respect to untreated cells. (B) LDH release in culture medium in control as well as in silenced neuroblastoma cells treated with Aβ1-42 oligomers for 24 h. In silenced cells oligomers failed to induce necrotic cell death after 12 h and 24 h of treatment. (C) LC3-II and SQSTM1 protein levels in cells with and without silencing for BECN1 and treated with oligomers of Aβ1-42 for 8 h and 12 h. The absence of BECN1 lowered the basal levels of both LC3 and SQSTM1 and treatment of cells with the oligomeric preparation of Aβ1-42 did not modify this trend. The error bars represent standard deviations. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 10. Primary cortical neuronal cultures treated with 1 μM Aβ1-42 monomers and oligomers confirm findings from SK-N-BE neuroblastoma cell lines on autophagy and apoptosis. (A) LC3 I-II protein levels in cells treated with Aβ1-42 monomers and oligomers at different time points. Monomeric preparations induce a significant increase in LC3-II protein levels that started at 1 h and was still observed after 48 h of incubation. Treatment of neuronal cells with oligomeric preparation produced a transient increase of LC3-II protein levels that was present after 6 h and 12 h, but not 24 h and 48 h. (B) CTSD activity in cortical cultures treated with Aβ1-42 monomers and oligomers up to 48 h. Only Aβ1-42 monomers significantly decreased CTSD activity about 40% to 60% between 6 and 48 h. (C) BACE1 activity in cortical cultures treated with Aβ1-42 monomers and oligomers. Monomers, but not oligomers, increase BACE1 activity of approximately 40% after 12 h to 48 h of treatment. (D) BCL2 and cleaved CASP3 protein levels in primary cortical cultures treated with Aβ1-42 monomers and oligomers. Oligomers, but not monomers, induce apoptotic cell death as confirmed by the significant decrease (-50%) of BCL2 and increase (+100 to 150%) of cleaved CASP3 protein levels. The error bars represent standard deviations. Experiments were conducted in triplicate. *, significantly different from controls (P < 0.05); **, significantly different from controls (P < 0.02).

Figure 11. Proposed effects on autophagy and apoptosis elicited by Aβ1-42 monomers and oligomers. (A) Monomers impede formation of the BCL2-BECN1 complex by activation of the MAPK/JNK pathway that phosphorylates BCL2. Monomers also cause early endosome accumulation due to an inhibitory action on lysosomal activity, resulting in intracellular accumulation of autophagosomes with elevation of levels of BECN1 and LC3-II, and inhibition of substrate degradation. (B) Oligomers favor the formation of the BCL2-BECN1 complex leading to apoptosis. They also cause a less profound increase in BECN1 and LC3-II levels and do not affect the autophagic flux.