The release of calcium from the endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway.

TLDR: It is demonstrated that the early Abeta- and PrP -induced perturbation of ER Ca2+ homeostasis affects mitochondrial function, activating the mitochondrial-mediated apoptotic pathway and help to clarify the mechanism implicated in neuronal death that occurs in AD and PrD.

About: This article is published in Neurobiology of Disease.The article was published on 2008-06-01 and is currently open access. It has received 202 citations till now. The article focuses on the topics: Inner mitochondrial membrane & Mitochondrion.

Figures

Fig. 6. Bax channel blocker prevents cytochrome c release induced by Aβ1– 40 and PrP106–126. Cortical neurons were treated for 6 h with 5 μMAβ1– 40, in the absence or in the presence of 50 nM of Bax channel blocker (A) or with 25 μM PrP106–126, in the absence or in the presence of 25 nM of Bax channel blocker (B). Cytochrome c levels in mitochondria- and cytosolicenriched fractions were detected as a 15-kDa band by Western blot analysis. Graphs represent the differences in expression levels from at least three different experiments, analyzed by densitometry. Data were expressed as the mean±SEM. ⁎pb0.05; ⁎⁎pb0.01 with respect to control values. #pb0.05; ##pb0.01 with respect to Aβ or PrP addition.

Fig. 7. Bax channel blocker reduces DNA fragmentation induced by Aβ1– 40 and PrP106–126. The number of cells exhibiting fragmented DNA was measured by fluorescence microscopy using SYTO-13 and PI staining in cortical neuronal cultures treated for 24 h with 0.5 μM Aβ1–40, in the absence or in the presence of 50 nM of Bax channel blocker (A) or with 25 μM PrP106–126, in the absence or in the presence of 25 nM of Bax channel blocker (B), as described in the Materials and methods section. The results, expressed as the percentage of the total number of cells, are the means±SEM of values corresponding at least to 3 experiments, each value being the mean of duplicate assays. ⁎pb0.05 with respect to control values. #pb0.05 with respect to Aβ or PrP addition.

Fig. 1. Aβ1–40 and PrP106–126 deplete ER Ca2+ content. The fluorescence ratio at 340/380 nm of the Ca2+-sensitive Fura-2/AM was monitored during 12 min, in the absence of external Ca2+. After 2 min, 2.5 μM thapsigargin was added to deplete ERCa2+. Representative F340/F380 traces of cortical neurons treated for 1 and 24 hwithAβ1–40 (0.5μM) (A) or with PrP106–126 (25 μM) (B) are presented. The peak amplitude of Fura-2 fluorescence was used to evaluate ER Ca2+ content and data from at least three different experiments, obtained from Aβ1–40- or PrP106–126-treated neurons, were normalized to the values determined in untreated control cells (C). Data were expressed as the mean±SEM. ⁎⁎⁎pb0.001 with respect to control values.

Fig. 8. Aβ1–40 and PrP106–126 induce an increase in the number of TUNEL-po with Aβ1–40 (0.5 μM) or PrP106–126 (25 μM), in the absence or in the presence Materials and methods section. Representative phase contrast, fluorescent (T magnification) are presented. (Graph) The numbers of TUNEL-positive cells are exp of values corresponding at least to 3 experiments. ⁎⁎⁎pb0.001 with respect to co

Fig. 2. Aβ1–40 and PrP106–126 reduceΔΨmit. Protection by antioxidants, IP3R an by the capacity of mitochondria from cortical neurons to take up the fluorescent cat (25 μM) or thapsigargin (2.5 μM), in the absence or in the presence of CoQ10 (1 μ (A) or 24 h (B). The results, expressed as the percentage of the baseline memb corresponding at least to 3 experiments, each value being the mean of duplicate ass ###pb0.001 with respect to Aβ, PrP or thapsigargin addition.

Fig. 4. GSH levels are increased in neurons treated with Aβ1–40 or PrP106–126. Protection by dantrolene and xestospongin. Cortical neurons were treated for 24 h with Aβ1–40 (0.5 μM) (A) or PrP106–126 (25 μM) (B), in the absence or in the presence of dantrolene (10 μM) or xestospongin C (1 μM). GSH content, expressed as percentage of control values, are the means±SEM of values corresponding at least to 3 experiments, each value being the mean of duplicate assays. ⁎pb0.05; ⁎⁎⁎pb0.001 with respect to control values. #pb0.05; ##pb0.01 with respect to Aβ or PrP addition.

Frequently asked questions

Q1. What is the role of cytochrome c in apoptosis?

Once released from mitochondria, cytochrome c binds to the apoptosis-inducing factor, mediating caspase-9 activation, which in turns activates caspase-3 and subsequently promotes apoptosis (Desagher and Martinou, 2000). 

Q2. What is the effect of thapsigargin on mitochondria?

The increment in ROS levels observed upon treatment with thapsigargin indicates that ER stress alters the equilibrium between ROS formation and the defence mechanisms of the cell. 

Q3. What is the effect of the synthetic A1–40 orPrP106–126?

In cortical neurons, addition of the synthetic Aβ1–40 orPrP106–126 peptides depletes ERCa2+ content, leading to cytosolicCa2 + overload. 

Q4. How many cells were plated on poly-L-lysine coated glass coverslips?

For the measurement of ROS levels and Rh123 retention, cells were plated on poly-L-lysine (0.1 mg/mL)-coated dishes at a density of 0.10×106 cells/cm2. 

Q5. What is the role of ER Ca2+ in the apoptosis pathway?

Accumulation of unfolded proteins due to the perturbation of ER Ca2+ homeostasis can activate the unfolded protein response (UPR) and, consequently, the ER stress-induced apoptosis pathway (Kaufman, 1999; Pashen, 2001). 

Q6. What is the role of Bcl-2 in apoptosis?

the authors have recently shown that overexpression of the anti-apoptotic Bcl-2 protein protects against Aβ and PrP toxicity (Ferreiro et al., 2007), further implicating Bcl-2 family proteins in apoptosis induced by these amyloidogenic peptides. 

Q7. What is the effect of the massive Ca2+ influx into mitochondria?

The massive Ca2+ influx into mitochondria collapse mitochondrial membrane potential Δψmit, leading to cell death (Duchen, 2000; Hajnóczky et al., 2000). 

Q8. What is the role of Bax in the release of apoptogenic factors?

a cytosolic Bcl-2 family protein, cantranslocate to mitochondria wherein it oligomerizes and inserts into the outer mitochondrial membrane, causing permeabilization and release of apoptogenic factors from the intermembrane space (Breckenridge and Xue, 2004). 

Q9. What was the method used for the identification of proteins of interest?

The identification of proteins of interest was facilitated by the usage of a prestained precision protein standard (Bio-Rad) which was run simultaneously. 

Q10. What is the role of ER Ca2+ in the depolarization of the mitochondrial?

Their data further demonstrate that Ca2+ released from the ER leads to the depletion of endogenous GSH levels and accumulation of reactive oxygen species, which were also involved in the depolarization of the mitochondrial membrane. 

Q11. What is the role of mitochondria in AD?

functional mitochondria were previously shown to be required for Aβ toxicity (Cardoso et al., 2001), revealing that mitochondria plays a fundamental role in the cell death that occurs in AD. 

Q12. What is the mechanism of ER Ca2+ release in mitochondria?

Their results demonstrate that, in cortical neurons, Aβ1–40 and PrP106–126 peptides activate the mitochondria-mediated apoptotic pathway, by a mechanism involving the release of Ca2+ from endoplasmic reticulum (ER) through channels associated with ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (IP3R). 

Q13. What is the effect of Fura-2 on the ER?

ER Ca2+ content was evaluated indirectly measuring Fura-2 fluorescence in the absence of external Ca2+, before and after the addition of thapsigargin, a SERCA-Ca2+ATPase inhibitor, which depletes ER stores. 

Q14. What is the effect of Bax channel blocker on apoptotic cells?

To ascertain that cytochrome c is released through the Bax channel formed in the outer mitochondrial membrane, the levels of this apoptogenic factor was analysed by Western blotting in mitochondrial and cytosolic fractions isolated from Aβ- or PrPtreated cortical neurons in the presence of a Bax channel blocker (Bombrun et al., 2003). 

Q15. What is the mechanism of the release of cytochrome c in cortical neurons?

The authors previously showed that both Aβ and PrP peptides induce the release of cytochrome c in cortical neurons both at 6 and 24 h (Ferreiro et al., 2006) and so the authors hypothesized that these peptides could induce the translocation of Bax to the mitochondria by a mechanism involving the release of Ca2+ from ER. 

Q16. What is the effect of the dye on mitochondrial membrane potential?

The changes in mitochondrial membrane potential (ψmit) were estimated using the fluorescent cationic dye Rh123, which accumulates in mitochondria as a direct function of the membrane potential and is released upon membrane depolarization (Palmeira et al., 1996, with some modifications). 

Q17. What is the effect of A and PrP on ER Ca2+ levels?

These results show that Aβ and PrP peptides affect ER Ca2+ homeostasis in cortical neurons, inducing a substantial reduction in ER Ca2+ content, which subsequently leads to the increase of cytosolic Ca2+ levels.