The first and second authors contributed equally.
Long-term Aβ exposure augments mCa2+-independent mROS-mediated depletion of cardiolipin for the shift of a lethal transient mitochondrial permeability transition to its permanent mode in NARP cybrids: a protective targeting of melatonin
Article first published online: 30 AUG 2012
© 2012 John Wiley & Sons A/S
Journal of Pineal Research
Volume 54, Issue 1, pages 107–125, January 2013
How to Cite
Hsiao, C.-W., Peng, T.-I., Peng, A. C., Reiter, R. J., Tanaka, M., Lai, Y.-K. and Jou, M.-J. (2013), Long-term Aβ exposure augments mCa2+-independent mROS-mediated depletion of cardiolipin for the shift of a lethal transient mitochondrial permeability transition to its permanent mode in NARP cybrids: a protective targeting of melatonin. Journal of Pineal Research, 54: 107–125. doi: 10.1111/jpi.12004
- Issue published online: 6 DEC 2012
- Article first published online: 30 AUG 2012
- Manuscript Accepted: 27 JUL 2012
- Manuscript Received: 27 JUN 2012
- CMRPD. Grant Number: 180491-3
- CMRPD. Grant Number: 170411-3
- CMRPG. Grant Number: 270341-2
- Chang Gung Medical Research Foundation
- amyloid β-peptide;
- mitochondrial reactive oxygen species;
- neurological muscle weakness, ataxia, and retinitis pigmentosa cybrids;
Mitochondrial dysfunction is a hallmark of amyloid β-peptide (Aβ)-induced neurodegeneration of Alzheimer's disease (AD). This study investigated whether mtDNA T8993G mutation-induced complex V inhibition, clinically associated with neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP), is a potential risk factor for AD and the pathological link for long-term exposure of Aβ-induced mitochondrial toxicity and apoptosis in NARP cybrids. Using noninvasive fluorescence probe–coupled laser scanning imaging microscopy and NARP cybrids harboring 98% mutant genes along with its parental 143B osteosarcoma cells, we demonstrated that Aβ-augmented mitochondrial Ca2+ (mCa2+)-independent mitochondrial reactive oxygen species (mROS) formation for a cardiolipin (CL, a major mitochondrial protective phospholipid)-dependent lethal modulation of the mitochondrial permeability transition (MPT). Aβ augmented not only the amount but also the propagation rate of mROS-induced mROS formation to significantly depolarize mitochondrial membrane potential (∆Ψm) and reduce mCa2+ stress. Aβ-augmented mROS oxidized and depleted CL, thereby enhances mitochondrial fission and movement retardation, which promoted the NARP-augmented lethal transient-MPT (t-MPT) to switch to its irreversible mode of permanent-MPT (p-MPT). Interestingly, melatonin, a multiple mitochondrial protector, markedly reduced Aβ-augmented mROS formation and therefore significantly reduced mROS-mediated depolarization of ∆Ψm, fission of mitochondria and retardation of mitochondrial movement to stabilize CL and hence the MPT. In the presence of melatonin, Aβ-promoted p-MPT was reversed to a protective t-MPT, which preserved ∆Ψm and lowered elevated mCa2+ to sublethal levels for an enhanced mCa2+-dependent O2 consumption. Thus, melatonin may potentially rescue AD patients associated with NARP symptoms.
Alzheimer's disease (AD), characterized by age-dependent decline in memory and cognitive function, first described by Alois Alzheimer in 1906, is a devastating neurodegeneration affecting nearly 30 million people worldwide nowadays. Although the precise pathological mechanism of AD is still controversial, the misfolding and aggregation of amyloid β-peptide (Aβ) and Aβ precursor proteins (AβPP) are considered as major causes of neurodegeneration for AD (for reviews, see [1, 2]). Relatively small portions of AD are results of gene mutation in particular molecules such as AβPP, presenilin 1, and presenilin 2 for early-onset AD (for review, see ) and apolipoprotein E gene allele 4 and sortilin-related receptor 1 gene for late-onset AD [4, 5]. Recently, new evidence has unveiled mitochondria as a novel and critical pathological target for Aβ and Aβ-related toxic molecules (for reviews, see [6-11]). Aβ and other toxic molecules are observed in the mitochondria of both the AD brain and transgenic mouse models of AD [12-15].
Aβ permeates the cellular membrane and lipid bilayer [16, 17] or is taken up by the TOM complex [18, 19] to enter the mitochondria. Aβ and AβPP, then, prudentially localize to the inner mitochondrial membranes and alter mitochondrial membrane integrity and function [19-22]. Aβ25–35 has been specifically shown to cause a rapid dose-dependent decrease in the activity of complex IV (cytochrome oxidase), while it has no effect on the activities of other enzymes tested . Recent data also indicate that Aβ may interact with other proteins such as Aβ-binding alcohol dehydrogenase in mitochondria and directly cause mitochondrial dysfunction [15, 24]. Aβ also alters the mitochondrial structural and functional dynamics ([25, 26]; for reviews, see [27, 28]) and results in dysregulation of mitochondrial trafficking and interorganellar communication, which can be crucial for mitochondrial quantity control and Aβ-induced neurodegeneration (for review, see ). Furthermore, disturbed Ca2+ homeostasis ([30-32]; for reviews, see [33-37]) accompanied by enhanced reactive oxygen species (ROS) ([38-41]; for review, see ) formation are found to be associated with AD . As a result, Aβ1–40 and Aβ25–35 [42, 43] trigger the mitochondrial permeability transition (MPT), a critical mechanism that releases mitochondrial proapoptotic factors [44, 45] for an enhanced mitochondria-mediated cell death and induction of critical pathological degeneration of neurons, synapses, and synaptic function (for reviews, see [11, 17, 46]). The MPT-dependent toxicity of Aβ is confirmed by a cyclophilin D (CypD) deficiency that attenuates mitochondrial and neuronal perturbations and improves learning and memory in AD (; for review, see ).
The precise cause and effect relationships, however, of how Aβ-induced stresses of mitochondrial Ca2+ (mCa2+) and mitochondrial reactive oxygen species (mROS) alter the activation of the MPT for final necrotic or apoptotic death of cells are still rather ambiguous. Using noninvasive fluorescence probe–coupled laser scanning imaging microscopy, this study revalues the individual pathological role for mCa2+ and mROS and how these stresses are associated mitochondrial dynamics of movement and fission during Aβ exposure, as well as how these factors interact for the final activation of the MPT. We proposed that cardiolipin (CL), a mitochondrial protective phospholipid [49, 50], is a critical potential link for Aβ-altered mCa2+ and/or mROS to the final modulation of the activity of the MPT. Precisely, dynamic modes of the protective transient-MPT (t-MPT) and the lethal permanent-MPT (p-MPT)  and their protection by melatonin, a multiple mitochondrial protector and MPT modulator [51, 52], were explored in detail during Aβ exposure. In addition, we challenged the positive role of mCa2+ in Aβ-induced mitochondrial dysfunction as long-term exposure to Aβ often results in significant depolarized mitochondrial membrane potential (∆Ψm), which reduces the essential driving force for the mitochondrial uniporter to take up Ca2+ from the cytosol during fluctuations or elevations of cytosolic Ca2+ and thus reduces the pathological role of mCa2+ in Aβ-mediated cell death.
Finally, the mtDNA defects of deletion and mutation are closely associated with AD ([53-57]; for reviews, see [58, 59]). For instance, the mtDNA ‘common deletion’, that is, the mtDNA 4977 bp deletion, increases 15-fold in AD brain . mtDNA A4336G, T414C and T477C mutations are found to be predominantly expressed in patients with AD compared with those in controls [56, 61]. Moreover, the technique using cytoplasmic hybridization cells (cybrids) of ρ0 (mtDNA less) cells fused with mtDNA from patients with AD show reduced cytochrome oxidase activity, elevated ROS, and reduced ATP levels [62-65]. This evidence thus suggests that mtDNA alteration–induced mitochondrial dysfunction may play crucial role in AD. In this study, we investigated specifically how mtDNA T8993G mutation influences the toxicity of Aβ. The mtDNA T8993G for Leu156Arg results in potent inhibition of the ATP synthase 6 of the F1F0-ATPases (the mitochondrial complex V), a complex that catalyzes the terminal step of the oxidative phosphorylation for ATP synthesis . Clinically, mtDNA T8993G is closely associated with syndromes of neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP), the so-called NARP mutation . To explore how mtDNA T8993G–induced complex V inhibition is a risk factor for the Aβ-induced neurodegeneration, NARP cybrids harboring 98% mtDNA T8993G mutation together with its parental 143B osteosarcoma cells  were conducted. NARP cybrids were used especially for investigating Aβ-induced mCa2+ stress as mitochondrial complex V inhibition enhances hyperpolarization of ∆Ψm. This mutation enhances mCa2+ stress, if there is any during Aβ exposure in NARP cybrids. Moreover, whether and how precisely a potent mitochondrial protector, melatonin [68, 69], reduces Aβ-induced mitochondrial stresses and their modulations on the MPT were explored [49-52, 70-72]. Specially, we investigated whether and how melatonin's effects on mitochondrial oxidative stress, CL oxidation, and depletion and how these alterations modulate mCa2+ levels, dynamic switching between the t-MPT (including the protective and lethal modes), and the lethal p-MPT during Aβ exposure. Lastly, we explored how melatonin alters mCa2+ to improve mitochondrial RC activity for ATP synthesis during long-term Aβ exposure in NARP cybrids.
Materials and methods
Establishment of NARP cybrids
The NARP cybrids were established as previously described . Briefly, skin fibroblasts obtained from a patient with Leigh's disease carrying the mtDNA T8993G mutation were enucleated and cytoplasmically fused with mtDNA less (ρ0) human osteosarcoma 143B cells. The NARP cybrids were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum supplemented with high glucose (4.5 g/mL), pyruvate (0.11 mg/mL), and uridine (0.1 mg/mL). NARP cybrids that have a mutant mtDNA ratio of approximately 98% were used for experiments, and comparisons were made to its parental 143B cells.
Chemicals and fluorescent dye loading for fluorescence measurement of mitochondrial events
All chemicals were obtained from Sigma (St. Louis, MO, USA), and fluorescent dyes were purchased from Molecular Probes Inc. (Eugene, OR, USA). Loading conditions for each specific fluorescent probe are described below: ROS was detected using 2 μm 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF); CL was detected using 80-nm nonyl acridine orange (NAO); MPT was detected using 1 μm calcein/AM, and 1 mm cobalt chloride (CoCl2) was added to quench cytosolic calcein; mCa2+ was detected using 2 μm rhod-2/AM; mitochondrial membrane potential was detected using 300 nm tetra-methylrhodamine methyl ester (TMRM). All fluorescent probes were loaded at room temperature for 30 min except TMRM that was loaded for 10 min to avoid its quenching effect. After loading, cells were rinsed three times with HEPES-buffered saline solution (containing 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 0.8 mm MgCl2, 10 mm glucose, and 10 mm HEPES, for a pH of 7.4). Dye-loaded cells were then mounted on a cell chamber for laser-coupled imaging microscopic observation. To investigate the protective effects of melatonin, we preincubated the cells with 100 μm melatonin 30 min ahead and during the exposure to Aβ25–35.
Quantification of ROS generation using a fluorescent spectrofluorimeter
The cells cultured in 96-well standard black/clear bottom plate (Greiner Bio-One International AG, Kremsmuenster, Austria). The ROS was detected with DCF (excitation and emission wavelengths were 490 and 520 nm, respectively). The fluorescent intensity of DCF was quantified on a Spectramax Gemini EM spectrofluorimeter (Molecular Devices, Sunnyvale, CA, USA). Results were analyzed by using SoftMax Pro 4.7 software (Molecular Devices) and Microsoft Excel software.
Imaging analysis of living cells
All confocal fluorescent images and image stacks were collected using a Zeiss LSM 510 META NLO mounted on an Axiovert 200 m inverted microscope (Carl Zeiss Microimaging Inc., Thornwood, NY, USA). All fluorescent images were collected using a Zeiss objective lens (Plan-Apochromat 100X, NA1.4 oil DIC M27). DCF, NAO, calcein and region of interest (ROI) were excited using the Argon/2 laser (30 mW) for excitation. The excitation wavelength was 488 nm, the main dichroic beam splitter was 488/561 nm, and the emission detection filter was band pass 500–550 nm. All the ROIs (3 × 3 μm2) were carried out by 100% 488 nm irradiation in 10 s. Rhod-2 and TMRM were excited using the DPSS laser (15 mW). The excitation wavelength was 561 nm, the main dichroic beam splitter was 488/561 nm, and the emission detection filter was long pass 575 nm. All images were processed and analyzed using MetaMorph software (Universal Imaging Corp., West Chester, PA, USA). Intensity levels were analyzed from the original images and graphed using Microsoft Excel software and Photoshop.
Measurement of mitochondrial movement and fission
Mitochondrial movement was measured from the overlapping mitochondrial area (in yellow; i.e., nonmoving area) of superimposed images of two consecutive images (one in red and the other in green) taken 2 min apart. Morphological change from thread like to fragmented shape was considered as mitochondrial fission. The average of ten representative populations of mitochondria in one single cell from 10 to 20 cells was calculated.
Measurement of cell viability
Cell viability was detected using the colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as previously described . Activity of a mitochondrial reductase to convert a soluble tetrazolium salt into an insoluble formazan precipitate was measured by an ELISA reader (A-5082; TECAN, Grödig/Salzburg, Austria). To investigate the protective effects of melatonin on Aβ-induced cytotoxicity, cells were pretreated with melatonin for at least 30 min before Aβ exposure. MTT assay was performed after 1 hr of Aβ exposure. Activity of mitochondrial reductase was calculated as the amount of MTT dye conversion relative to the changes seen in sham-treated control cells. Data were represented as mean ± S.E. of at least three independent experiments.
Measurement of cellular oxygen consumption
Cells (5 × 105/100 μL) after trypsinization were immediately transferred to the chamber of Mitocell equipped with a Clark oxygen electrode (782 Oxygen Meter; Strathkelvin Instrument, Glasgow, UK) for measurement of cellular O2 consumption as previously described . The rate of O2 consumption was calculated from the slopes and expressed in % of O2 consumed per min per 5 × 105 cells.
The results are expressed as mean ± S.E.M., and statistical significance was evaluated by either one-way or multi-factorial analysis of variance (ANOVA). A value of P < 0.05 was considered significant. Each experiment was repeated at least three times.
The effects of NARP-induced inhibition of the mitochondrial complex V on mROS formation and ∆Ψm alteration (see also ) were simultaneously and continuously detected using DCF and TMRM, respectively, at rest and during an oxidative stress induced by exogenous administration of H2O2 (Fig. 1). Fig. 1A,B demonstrate that NARP mutation resulted in high resting levels of ROS (Fig. 1A,A′) with hyperpolarized ∆Ψm (Fig. 1B,B′) in NARP cybrids compared with the parental 143B cells. The NARP-augmented ROS formation was enhanced further during an oxidative stress by H2O2 (10 mm) exposure. Thus, NARP mutation may enhance mROS-induced mROS formation (Fig. 1A). The ∆Ψm in NARP cybrids, although more hyperpolarized at rest, depolarized much faster than that in 143B cells during H2O2 exposure, suggesting NARP mutation-induced hyperpolarization of ∆Ψm was vulnerable to the oxidative insult (Fig. 1B,B′). Dose dependency of H2O2-induced ROS formation (Fig. 1C) and ∆Ψm depolarization (Fig. 1D) in NARP cybrids was observed. Higher levels of ROS were followed by faster ∆Ψm depolarization in increased concentrations of H2O2 suggesting ROS-induced ∆Ψm depolarization.
Next, the effects of how long-term (up to 24 hr) accumulation of Aβ (20–50 μm) on ROS (Fig. 1E) and ∆Ψm (Fig. 1F) at rest as well as during an oxidative stress induced by H2O2 (10 mm) were explored in NARP cybrids. Interestingly, Aβ dose-dependently elevated the resting levels of ROS (Fig. 1E,E′). Aβ-elevated ROS levels were enhanced further during a secondary oxidative stress by H2O2 (10 mm) exposure (Fig. 1E). Correspondingly, Aβ also dose-dependently depolarized resting ∆Ψm (Fig. 1F′). Aβ-depolarized ∆Ψm was enhanced further during H2O2 (10 mm) exposure (Fig. 1F). In high concentrations of H2O2 (>50 mm), cell viability was compromised significantly in NARP cybrids (not shown). These results, thus, indicate that the accumulation of Aβ, similar to an oxidative insult, may sensitize ROS-induced ROS formation and the depolarization of ∆Ψm in NARP cybrids. Both Aβ-enhanced ROS and the depolarized ∆Ψm are key components in the activation of the MPT. The lag of ∆Ψm depolarization (2–5 min) to ROS formation (instantaneously) during H2O2 exposure also suggests that ∆Ψm depolarization is secondary to Aβ-induced ROS formation in NARP cybrids. As ∆Ψm is also the main driving force for mCa2+ uptake, we propose that Aβ exposure reduces mCa2+ stress in NARP cybrids (see also Fig. 3).
As a result of Aβ toxicity, long-term exposure of Aβ alone compromised cell viability significantly in NARP cybrids (Fig. 2A). After Aβ exposure (20 hr, 50 μm), cell viability decreased to 90% and <30% in 143B cells and NARP cybrids, respectively (see Fig. 2A). Interestingly, a potent mitochondrial protector and an antioxidant, melatonin (100 μm) (see [49-52, 72]), significantly reduced the Aβ-induced cell death in NARP cybrids (Fig. 2A), suggesting that mitochondria-targeted oxidative stress may play a significant role in Aβ-induced toxicity and cell death in NARP cybrids; This is supported by the Aβ-induced time-dependent ROS formation during 20-hr Aβ incubation in NARP cybrids (Fig. 2B). Melatonin significantly reduced Aβ-induced dose-dependent (20 and 50 μm) elevation of ROS in NARP cybrids (Fig. 2C).
We next investigated how Aβ-induced ROS formation altered the levels of mCa2+, another potent mitochondrial stress that activates the MPT, and CL, a critical mitochondrial protective phospholipid. Resting levels of mCa2+ and CL were simultaneously imaged using rhod-2 and NAO, respectively, before and after Aβ incubation (50 μm, 12 hr) in 143B cells and NARP cybrids (Fig. 3). Compared with 143B cells, the resting level of mCa2+ in NARP cybrids was higher due possibly to the inhibition of complex V-induced hyperpolarization of ∆Ψm (Fig. 3A,A′, see also ). Aβ did not alter the resting level of mCa2+ in 143B cells, however, it significantly reduced the resting level of mCa2+ in NARP cybrids. These data indicate that Aβ-induced depolarization of ∆Ψm significantly reduces the mitochondrial driving force for mCa2+ uptake and that mCa2+ may play a minor role in Aβ-induced mitochondrial stress in NARP cybrids. Interestingly, melatonin (100 μm) elevated Aβ-reduced mCa2+ to a level lower than that of the control in NARP cybrids. Whether or not melatonin-elevated mCa2+ enhances or reduces Aβ-induced mitochondrial stress is considered in Fig. 9.
In the meantime, long-term Aβ exposure (50 μm, 12 hr) altered the CL distribution in 143B cells slightly. CL is distributed evenly in the thread-like mitochondria before and after Aβ exposure in 143B cells. In contrast, long-term Aβ exposure depleted CL significantly (>50%) from the already-fragmented (fissed) mitochondria in NARP cybrids (Fig. 3B,B′; see also Fig. 5 and ). Aβ exposure also resulted in further fragmentation of mitochondria in NARP cybrids. Interestingly, melatonin (100 μm) significantly protected against Aβ-induced depletion of CL and fragmentation of mitochondria in NARP cybrids.
To explore how Aβ (50 μm, 12 hr) influenced mitochondrial dynamics including mitochondrial movement and morphology, we loaded the cells with TMRM, and mitochondrial dynamics were analyzed using time-lapse imaging continuously both with and without Aβ in 143B cells and NARP cybrids. NARP-induced significant alterations in mitochondrial dynamics including retardation of mitochondrial movement (Fig. 4) and mitochondrial fission (Fig. 5) in NARP cybrids (see also ). The nonmoving mitochondrial population analyzed from the overlapping area (orange area) of two consecutive images (red and green images) are 43 ± 4% and 55 ± 6% in 143B cells and NARP cybrids, respectively. This suggests that the NARP mutation–induced complex V inhibition alters the mitochondrial moving machinery (see also ). Aβ (50 μm, 12 hr) further enhanced the retardation of mitochondrial movement to 61 ± 6% and 86 ± 3% in 143B cells and NARP cybrids, respectively. Interestingly, melatonin (100 μm) significantly reduced Aβ-induced retardation of mitochondrial movement in NARP cybrids. Quantitative analysis of Aβ-induced retardation of mitochondrial movement is shown in Fig. 4B.
Figure 5 demonstrates how Aβ altered mitochondrial morphology. NARP mutation induced a significant fragmentation compared with that of 143B cells of the mitochondria in NARP cybrids at rest. Long-term exposure of Aβ (50 μm) also induced a significant fission of the mitochondria in 143B cells and NARP cybrids. Aβ also caused swelling of the mitochondria in NARP cybrids (Fig. 5A). Melatonin markedly reduced Aβ-induced fission of the mitochondria in NARP cybrids. Quantitative analysis of mitochondrial fission is shown in Fig. 5B.
Next, we explored how long-term Aβ exposure–induced mitochondrial stresses of ROS formation, ∆Ψm depolarization, and CL depletion with enhanced fission of mitochondria altered the stability of the MPT at rest by continuously imaging for 20 min the activity of the MPT (including t-MPT and p-MPT) using calcein (in the presence of Co2+) and TMRM (Fig. 6). The loss of the calcein signal without a reduction in the TMRM signal was considered as the appearance of the t-MPT. The recording stresses induced by illuminating 488 and 561 nm lasers were measured and compared. Fig. 6A,A′ (∆Ψm/TMRM) demonstrated that the recording lights had no effect on ∆Ψm in 143B cells and NARP cybrids. Aβ depolarized slightly the ∆Ψm in 143B cells. In contrast, Aβ depolarized significantly the ∆Ψm in NARP cybrids. NARP-augmented hyperpolarization of ∆Ψm dropped significantly from the highest level to the lowest one after Aβ exposure (Fig. 6A′). Note the TMRM signal was reduced further during the continuous recording time period in Aβ-exposed NARP cybrids suggesting they were more vulnerable to the stress of recording lasers illumination. Interestingly, melatonin provided potent prevention against Aβ-induced depolarization of ∆Ψm in NARP cybrids. Melatonin also prevented significantly recording laser-induced further depolarization of ∆Ψm in Aβ-exposed NARP cybrids.
Figure 6B,B′ demonstrate that the activity of the MPT remained rather constant before and after long-term Aβ exposure during the 20-min recording period in 143B cells. In contrast, the MPT in NARP cybrids was rather unstable even before Aβ exposure because it opened about 14 min after the recording and gradually lost up to 42% of calcein signal at the end of recording. Aβ exposure caused a further significant loss of the calcein signals at rest (40%) suggesting a high activity of the MPT opening in Aβ-treated NARP cybrids. The calcein signal continuously decreased during the 20 min of recording (35%) in Aβ-exposed NARP cybrids (Fig. 6B′).
The superimposed images (Fig. 6C) and quantitatively analyzed curves (Fig. 6D–H) of TMRM and calcein demonstrate the degree of t-MPT in each condition in 143B cells and NARP cybrids. In the control, the persistent orange mitochondria in TMRM/calcein overlay images throughout the 20-min recording time suggests that there was no t-MPT in 143B cells (see also Fig. 6D). Aβ exposure also did not trigger the t-MPT but resulted in slight depolarization of ∆Ψm (decrease in red) in 143B cells; thus, the color of superimposed images shifted from orange to yellowish (Fig. 6E). In contrast, NARP mitochondria show significant t-MPT (red mitochondria in the TMRM/calcein overlay images, that is, more TMRM signal than calcein signal) at the beginning of the recording. The t-MPT seen in NARP cybrids was more likely a lethal t-MPT as the illumination light enhanced further the loss of calcein after 14 min of recording (Fig. 6F; see also Figs 7 and 8). Aβ exposure in NARP cybrids not only greatly depolarized ∆Ψm but also caused major loss of calcein signal suggesting the shifting of NARP-augmented t-MPT to p-MPT in NARP cybrids (Fig. 6G). Note that the more fragmented mitochondria contained more depolarized ∆Ψm (less TMRM) and MPT opening (less calcein) for an enhanced p-MPT indicating fission of the mitochondria-enhanced Aβ toxicity on the MPT (Fig. 6G).
Finally, melatonin effectively prevented not only Aβ-induced loss of ∆Ψm but also the MPT opening. The signals of TMRM and calcein both increased significantly in melatonin-treated Aβ-exposed NARP cybrids. Interestingly, melatonin-preserved calcein signal decreased at about 16 min after the recording, suggesting that melatonin reversed Aβ-induced shift of p-MPT to t-MPT (red mitochondria in TMRM/calcein overlay images at 20 min, Fig. 6H). Whether melatonin-preserved t-MPT is protective is considered in Figs 7 and 8. Individual effects on ∆Ψm and calcein in both cells during various conditions are summarized in Fig. 6A″,B″. NARP mutation augmented Aβ-induced ∆Ψm depolarization and MPT opening, and melatonin effectively rescued the Aβ-induced ∆Ψm depolarization and MPT opening in NARP cybrids.
To document the lethality of NARP-induced t-MPT and the toxicity of Aβ on the MPT, the dynamic modulation of the MPT was analyzed using time lapse imaging at rest and during a secondary mitochondrial oxidative stress induced by ROI irradiation using 488 nm laser with and without the exposure to Aβ in NARP cybrids (Fig. 7). The 488-nm irradiation-induced secondary mitochondrial oxidative stress dramatically depolarized ∆Ψm. This was greatly enhanced by the Aβ exposure. Melatonin significantly improved resting ∆Ψm and prevented stress of ROI-induced ∆Ψm depolarization in Aβ-treated NARP cybrids (Fig. 7A, D and D′). Simultaneously, the pore opening was enhanced in the presence of Aβ at rest (Fig. 7B, E and E′) and ROI-induced mitochondrial oxidative stress triggered further the Aβ-induced opening of the MPT (mitochondria lost calcein significantly). Again, melatonin significantly restricted the opening of MPT at rest and during ROI-induced oxidative stress in Aβ-exposed NARP cybrids (Fig. 7E).
The TMRM/calcein overlay images in Fig. 7C (see also analyzed curves in Fig. 7F) show that t-MPT occurred before and after mitochondrial were exposed to ROI-induced oxidative stress. But the t-MPT soon shifted to p-MPT at 4 min after the ROI stress in NARP cybrids suggesting the t-MPT at rest in NARP cybrids is rather lethal. After long-term exposure to Aβ, the t-MPT reduced significantly (no red signal in overlay images), and the p-MPT (mitochondria lost both green and red signals) occurred immediately after the ROI-induced oxidative stress indicating that Aβ shifted the NARP-induced lethal t-MPT to p-MPT in NARP cybrids (Fig. 7G). Interestingly, in the presence of melatonin, the MPT pore was much resistant to ROI stress, and t-MPT did not occur until 10–12 min after the ROI stress (Fig. 7H).
To further confirm that NARP-induced t-MPT and Aβ-induced p-MPT are both lethal and melatonin-preserved t-MPT is protective, we measured simultaneously two major mitochondrial stresses mCa2+ and mROS using rhod-2 and DCF, respectively, using the same secondary oxidative stress induced by 488 nm laser ROI in NARP cybrids, Aβ-exposed NARP cybrids, and Aβ-exposed NARP cybrids in the presence of melatonin (Fig. 8). ROI stress enhanced significantly mCa2+ stress in NARP cybrids (Fig. 8A,D). ROI-induced increase in mCa2+, however, was not observed in Aβ-exposed NARP cybrids suggesting that long-term exposure to Aβ resulted in minor mCa2+ stress because of Aβ-induced depolarization of ∆Ψm (Figs 1 and 6). Interestingly, melatonin-increased ROI-induced mCa2+ to a sublethal level (see Fig. 9) possibly because the ∆Ψm was higher than that in the Aβ-exposed NARP cybrids.
The ROI stress simultaneously and significantly induced ROS formation particularly in the mitochondria area (mROS) in NARP cybrids, which was enhanced in the Aβ-exposed NARP cybrids. Melatonin significantly limited ROI-induced mROS formation in Aβ-exposed NARP cybrids (Fig. 8B,E). The rhod-2/DCF overlay images of mCa2+ and mROS demonstrated a marked correlation between these two stresses during ROI stress in NARP cybrids (Fig. 8C,F–H). Interestingly, only minor mCa2+ but vigorous mROS stress were observed in Aβ-exposed NARP cybrids. Note the cell blebbing and death after 14 min of ROI stress as indicated by the loss of DCF signal. The low signal of rhod-2 and DCF in the melatonin-treated Aβ-exposed NARP cybrids indicates that melatonin significantly reduced both mitochondrial stresses. Aβ significantly enhanced the amplitude and rate of propagation of mROS after the stress of ROI laser irradiation was applied (Fig. 8I).
Finally, we measured how long-term exposure to Aβ-altered mCa2+ may influence Ca2+-dependent O2 consumption in 143B cells and NARP cybrids and how melatonin protects them (Fig. 9). Compared with 143B cells, NARP mutation significantly decreased their O2 consumption. Long-term exposure to Aβ decreased further lowered O2 consumption in both 143B cells and NARP cybrids. Melatonin-elevated O2 consumption suggesting that the melatonin-increased mCa2+ level is under the sublethal level for stimulating O2 consumption during Aβ exposure.
This study demonstrates for the first time Aβ-augmented potent mCa2+-independent mROS formation and its lethal modulation on the MPT as a major pathological toxicity for long-term exposure of Aβ-induced cell death in NARP cybrids. In addition, our results suggest that mtDNA T8993G mutation-induced inhibition of mitochondrial complex V is a potential risk factor in augmenting Aβ-induced mitochondrial toxicity in patients with AD. Long-term exposure to Aβ sensitized mROS-induced mROS formation not only in the quantity of mROS formed but also in the rate of mROS propagation. Unlike acute Aβ toxicity, long-term Aβ exposure significantly depolarized ∆Ψm. As ∆Ψm is the major driving force for mCa2+ uniporter to take up Ca2+, Aβ-depolarized ∆Ψm reduced greatly the pathological toxicity of mCa2+ in Aβ-induced cell death. Aβ-augmented mCa2+-independent mROS formation resulted in significant oxidation and hence depletion of CL, a major mitochondrial protective phospholipid. Aβ also caused significant fission of mitochondria and retardation of mitochondrial movement. These Aβ-augmented mROS-mediated mitochondrial stresses and possibly Aβ itself promoted the NARP-augmented lethal t-MPT to anchor at p-MPT for final apoptotic death. Interestingly, melatonin, a multiple mitochondrial protector, markedly scavenged Aβ-augmented mROS formation and hence reduced significantly mROS-mediated stress to stabilize the MPT by reversing Aβ-promoted p-MPT back to its protective mode of t-MPT. Melatonin-preserved ∆Ψm recovered mCa2+ uptake, and the protective t-MPT allowed the overloaded mCa2+ to be released from the mitochondria and maintained at sublethal levels for an enhanced mitochondrial O2 consumption and ATP formation. Because of this unique multiple protective targeting, we conclude melatonin is a potential drug for the treatment of AD patients with NARP syndromes. Precise schematic illustration of Aβ-augmented mCa2+-independent mROS-mediated mitochondrial stress and its lethal modulation on the MPT and protections by melatonin in NARP cybrids is shown in Fig. 10.
Dysregulated mCa2+ homeostasis is crucial in the pathogenesis of neurodegenerative diseases by triggering cyclosporine A/CypD sensitive MPT for lethal proteins release to cumulate apoptosis . Although Aβ-disturbed Ca2+ signaling leads to degeneration of neuron (for reviews, see [35, 75-79]), whether mCa2+ stress plays a role in Aβ-induced mitochondrial dysfunction is still under debate . In general, exposure to Aβ (1–40 or 1–42) results in an elevation of the resting cytosolic Ca2+ [35, 81] because Aβ oligomers form Ca2+-permeable pores in the plasma membrane . Studies of patients with AD, animal models of AD, and AD cybrids also support for the disruption of neuronal Ca2+ regulation in the neurotoxic action of Aβ [38, 77]. However, to generate mCa2+ stress, mitochondria require normal ∆Ψm as the driving force for the mCa2+ uniporter to uptake Ca2+ from the cytosol (for review, see ). Thus, whether Aβ exposure influences ∆Ψm is crucial for establishing Aβ-induced mCa2+ stress. In this study, we demonstrate that Aβ dose-dependently depolarized ∆Ψm, especially after long-term (>12 hr) exposure. The depolarization is enhanced further if Aβ-exposed cells receive secondary stress such as mitochondrial oxidative stress either by H2O2 or by ROI visible laser irradiation. As a result, Aβ significantly reduces the driving force for mCa2+ uptake and therefore minimizes the toxic effects of mCa2+. This is supported by the evidence that ∆Ψm in mitochondria of aged cerebellar granular neurons are depolarized and less efficient in handling Ca2+ load . Cortical mitochondria from 12-month-old mice show a reduced capacity for Ca2+ uptake when changed with CaCl2 pulse, compared with those of 6-month-old mice . Additionally, most studies that observed Aβ-induced mCa2+ stress and toxicity were carried out during an acute exposure of Aβ at where the ∆Ψm was not disturbed significantly [30, 46, 81, 84]. It was also observed that the Aβ1–42 oligomer-induced mCa2+ overload is limited to a pool of mitochondria that are located close to the site of Ca2+ entry and release . A series of nonsteroidal anti-inflammatory drugs including salicylate, sulindac sulfide, indomethacin, ibuprofen, and R-flurbiprofen depolarize mitochondria prevent mitochondrial Ca2+ overload, cytochrome c release, and cell death induced by Aβ oligomers . Additionally, the Ca2+ storage capacity of rat brain mitochondria declines during the postnatal development without a change in ROS production capacity . In the study in which Aβ stimulated ryanodine receptor-mediated Ca2+ release, N-acetylcysteine, an antioxidant, completely blocked the emergence of Ca2+ signals induced by Aβ . The evidence including that from this study thus suggests strongly that Aβ-augmented mROS play a primary role and are crucial for the long-term Aβ-induced mitochondrial pathologies and apoptosis in NARP cybrids. Interestingly, melatonin, as a potent mitochondrial antioxidant , potently scavenges Aβ-augmented mROS formation and hence reduces significantly mROS-mediated multiple stresses to stabilize the MPT by reversing Aβ-promoted p-MPT back to its protective mode of t-MPT. Melatonin-preserved ∆Ψm recovers mCa2+ uniporter for mCa2+ uptake. To reduce mCa2+ overload, the melatonin-preserved protective t-MPT allows the overloaded mCa2+ to be released from the mitochondria and maintained at sublethal levels for an enhanced mitochondrial O2 consumption and ATP formation due possibly to the activation of mCa2+-dependent dehydrogenases (for review, see ).
Interestingly, Aβ-induced mROS stress results in multiple and fatal augmentation of mitochondrial pathologies in NARP cybrids associated with mtDNA T8993G mutation-induced complex V inhibition suggesting that mtDNA T8993G mutation is a potential risk factor for the enhancement of Aβ-induced mitochondrial toxicity and neurodegeneration in patients with AD. In fact, we observed that mtDNA-augmented mROS formation with enhanced apoptosis in common deletion (mtDNA 4977 bp deleted) cybrids [49, 87] and in RBA-1 astrocytes containing defective mitochondrial complex I because of long-term rotenone exposure (RT-RBA-1 cells, unpublished data) suggesting that mtDNA mutations or complex defects may potentially enhance neurodegenerative disorders including AD.
The Aβ-augmented mROS is potent and lethal because it augments not only the amount of mROS formed but also the rate of mROS propagating among the mitochondrial populations in NARP cybrids. This is because Aβ-induced mROS (i) depolarizes ∆Ψm, (ii) depletes CL, (iii) induces fission and retardation of the mitochondria, and (iv) promotes the p-MPT. Although mROS can target these events directly and separately, synergistic effects amount these stresses significantly and viciously augment each event. Aβ enhances fragmentation (fission) of mitochondria decreases mitochondrial resistance to apoptotic insults (for review, see [88, 89]). Although, fission of mitochondria forms a protective ‘firewall’ to reduce propagation of mROS for preconditioning protection , the Aβ (this study)- as well as mtDNA T8993G mutation ()- and mtDNA 4977 bp deletion ([49, 87])-induced fission appear to be rather fatal. This is possibly because fission of mitochondria disrupts the supercomplexes of ‘respirasomes’  to increase electron leakage and hence mROS formation. Aβ- and NARP-augmented fission and retardation of mitochondrial movement and fragmentation of mitochondria  can be prevented by the administration of melatonin presumably via its potent anti-mitochondrial oxidative effects confirms that mROS are the primary cause for the alteration in mitochondrial dynamics. This is obvious because the antioxidant N-acetylcysteine also prevents the mitochondrial fragmentation induced by Aβ . These results, therefore, imply that the interaction of Aβ with fission machinery can be crucial in mitochondrial pathogenesis during neurodegeneration and may serve as therapeutic target for the treatment of AD.
Mitochondrial reactive oxygen species formation–induced mitochondrial fission and retardation of mitochondrial movement often are associated with the depletion of CL. Also, the depletion of CL often precedes fission as CL is the major mitochondrial protective phospholipid that makes up more than 30% of the mitochondrial lipid; oxidation and depletion of CL can affect directly the stability of both respirasome complexes and the MPT, which augments electron leakage for an enhanced ROS formation to further harm mitochondrial dynamics. Our previous studies showed that mROS-mediated CL depletion is associated with fissed mitochondria obtained from mtDNA 4977 bp deletion cybrids [49, 87] and from RBA-1 astrocytes containing defected mitochondrial complex I because of long-term rotenone exposure (unpublished data) and can be significantly reduced by melatonin, a potent mitochondria-targeted antioxidant [49, 50, 69, 87]. This is consistent with mROS-mediated CL depletion and its associated alterations of mitochondrial dynamics of fission and movement retardation greatly enhance neurodegeneration including AD.
Aβ-induced mROS formation results in depolarization of ∆Ψm, which not only reduces energy production but also impacts negatively on the modulation of the MPT. Conversely, overloaded mCa2+ often initiates protective t-MPT, and mROS promote the p-MPT without the occurrence of the t-MPT, which is crucial in reducing mitochondrial overloaded toxins from maintaining ∆Ψm for energy production . Aβ-induced mCa2+-independent mROS formation triggered p-MPT viciously enhances more mROS formation, that is, mROS-induced mROS formation ([93, 94]; for review, see ) and irreversibly leads cells to the ‘point of no return’ for apoptotic or necrotic death. Aβ may greatly enhance the sensitive of CypD, a mCa2+ and ROS sensitivity mPTP components, to mROS due possibly to the modulation by Aβ itself or by Aβ-enhanced mROS-mediated depletion of CL and fission of the mitochondria . Thus, early termination of the Aβ-induced mROS formation as well as mROS-mediated p-MPT by melatonin may be crucial and essential in minimizing in Aβ-induced mitochondrial pathologies in AD treatment.
This work was supported by the grants CMRPD 180491-3 and CMRPD 170411-3 (to Jou) and CMRPG 270341-2 (to Peng) from the Chang Gung Medical Research Foundation, Taiwan.
- 1Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol 2007; 8:101–112., .
- 2Alzheimer's disease: pathological mechanisms and the beneficial role of melatonin. J Pineal Res 2012; 52:167–202., , et al.
- 3Mapping cellular transcriptosomes in autopsied Alzheimer's disease subjects and relevant animal models. Neurobiol Aging 2006; 27:1060–1077., .
- 4Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease. Neurology 1993; 43:1467–1472., , et al.
- 5The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease. Nat Genet 2007; 39:168–177., , et al.
- 6Alzheimer's disease: a lesson from mitochondrial dysfunction. Antioxid Redox Signal 2007; 9:1621–1630., , .
- 7Mitochondrial cascade hypothesis of Alzheimer's disease: myth or reality? Antioxid Redox Signal 2007; 9:1631–1646., , , .
- 8Mitochondrial dysfunction in aging and Alzheimer's disease: strategies to protect neurons. Antioxid Redox Signal 2007; 9:1647–1658..
- 9Mitochondrial dysfunction: the first domino in brain aging and Alzheimer's disease? Antioxid Redox Signal 2007; 9:1659–1675., , et al.
- 10A amyloid-beta interaction with mitochondria. Int J Alzheimers Dis 2011; 2011:925050., .
- 11Alzheimer's disease: effects of beta-amyloid on mitochondria. Mitochondrion 2011; 11:13–21., , .
- 12Amyloid-beta and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice. Proc Natl Acad Sci USA 2009; 106:20057–20062., , et al.
- 13Oligomeric and fibrillar species of beta-amyloid (A beta 42) both impair mitochondrial function in P301L tau transgenic mice. J Mol Med (Berl) 2008; 86:1255–1267., , et al.
- 14Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice. Neurobiol Aging 2009; 30:1574–1586., , et al.
- 15ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's disease. Science 2004; 304:448–452., , et al.
- 16Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis. Neurology 2006; 66:S74–S78., .
- 17Amyloid beta, mitochondrial dysfunction and synaptic damage: implications for cognitive decline in aging and Alzheimer's disease. Trends Mol Med 2008; 14:45–53., .
- 18Mitochondria and Alzheimer's disease: amyloid-beta peptide uptake and degradation by the presequence protease, hPreP. J Bioenerg Biomembr 2009; 41:447–451., , .
- 19The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae. Proc Natl Acad Sci USA 2008; 105:13145–13150., , et al.
- 20Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain. J Biol Chem 2008; 283:9089–9100., , , , .
- 21Mitochondrial abnormalities in Alzheimer's disease. J Neurosci 2001; 21:3017–3023., , et al.
- 22The spirostenol (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate blocks mitochondrial uptake of Abeta in neuronal cells and prevents Abeta-induced impairment of mitochondrial function. Steroids 2006; 71:725–735., , , , .
- 23Beta-Amyloid fragment 25–35 selectively decreases complex IV activity in isolated mitochondria. FEBS Lett 1999; 457:131–134., , .
- 24ABAD enhances Abeta-induced cell stress via mitochondrial dysfunction. FASEB J 2005; 19:597–598., , et al.
- 25Membrane integrity and amyloid cytotoxicity: a model study involving mitochondria and lysozyme fibrillation products. J Mol Biol 2011; 409:826–838., , .
- 26Melatonin treatment restores mitochondrial function in Alzheimer's mice: a mitochondrial protective role of melatonin membrane receptor signaling. J Pineal Res 2011; 51:75–86., , et al.
- 27Energetics in the pathogenesis of neurodegenerative diseases. Trends Neurosci 2000; 23:298–304..
- 28Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 2006; 443:787–795., .
- 29Mitochondria: the next (neurode) generation. Neuron 2011; 70:1033–1053., .
- 30Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs. PLoS ONE 2008; 3:e2718., , , , .
- 31Amyloid beta oligomers induce Ca2+ dysregulation and neuronal death through activation of ionotropic glutamate receptors. Cell Calcium 2010; 47:264–272., , et al.
- 32Amyloid beta-peptide oligomers stimulate RyR-mediated Ca2+ release inducing mitochondrial fragmentation in hippocampal neurons and prevent RyR-mediated dendritic spine remodeling produced by BDNF. Antioxid Redox Signal 2011; 14:1209–1223., , et al.
- 33The importance of being subtle: small changes in calcium homeostasis control cognitive decline in normal aging. Aging Cell 2007; 6:267–273., .
- 34Mitochondria in neuroplasticity and neurological disorders. Neuron 2008; 60:748–766., , .
- 35Aberrant subcellular neuronal calcium regulation in aging and Alzheimer's disease. Biochim Biophys Acta 2011; 1813:965–973., .
- 36Neuronal calcium homeostasis and dysregulation. Antioxid Redox Signal 2011; 14:1261–1273., .
- 37Calcium signaling and amyloid toxicity in Alzheimer disease. J Biol Chem 2010; 285:12463–12468., , .
- 38Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer's disease. J Neurosci 1997; 17:4612–4622., , et al.
- 39Mitochondrial network determines intracellular ROS dynamics and sensitivity to oxidative stress through switching inter-mitochondrial messengers. PLoS ONE 2011; 6:e23211., , .
- 40Prolonged exposure of cortical neurons to oligomeric amyloid-beta impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (-)-epigallocatechin-3-gallate. ASN Neuro 2011; 3:e00050., , et al.
- 41Abeta oligomers induce neuronal oxidative stress through an N-methyl-d-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. J Biol Chem 2007; 282:11590–11601., , et al.
- 42Amyloid beta-peptide promotes permeability transition pore in brain mitochondria. Biosci Rep 2001; 21:789–800., , , .
- 43Effect of amyloid beta-peptide on permeability transition pore: a comparative study. J Neurosci Res 2002; 69:257–267., , , , .
- 44Abeta (31–35) and Abeta (25–35) fragments of amyloid beta-protein induce cellular death through apoptotic signals: role of the redox state of methionine-35. FEBS Lett 2005; 579:2913–2918., , , , , .
- 45Amyloid beta peptide induces cytochrome C release from isolated mitochondria. NeuroReport 2002; 13:1989–1993., , et al.
- 46Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease. Exp Neurol 2009; 218:286–292..
- 47Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease. Nat Med 2008; 14:1097–1105., , et al.
- 48Mitochondrial permeability transition pore in Alzheimer's disease: cyclophilin D and amyloid beta. Biochim Biophys Acta 2010; 1802:198–204., .
- 49Melatonin protects against common deletion of mitochondrial DNA-augmented mitochondrial oxidative stress and apoptosis. J Pineal Res 2007; 43:389–403., , et al.
- 50mtDNA T8993G mutation-induced mitochondrial complex V inhibition augments cardiolipin-dependent alterations in mitochondrial dynamics during oxidative, Ca2+, and lipid insults in NARP cybrids: a potential therapeutic target for melatonin. J Pineal Res 2012; 52:93–106., , , , , .
- 51Melatonin preserves the transient mitochondrial permeability transition for protection during mitochondrial Ca2+ stress in astrocyte. J Pineal Res 2011; 50:427–435..
- 52Visualization of melatonin's multiple mitochondrial levels of protection against mitochondrial Ca2+-mediated permeability transition and beyond in rat brain astrocytes. J Pineal Res 2010; 48:20–38., , et al.
- 53High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer's disease brain. Hum Mol Genet 2002; 11:133–145., , , , .
- 54Mitochondrial genomic contribution to mitochondrial dysfunction in Alzheimer's disease. Int J Alzheimers Dis 2006; 9:183–193., , , , , .
- 55Are mitochondria critical in the pathogenesis of Alzheimer's disease? Brain Res Brain Res Rev 2005; 49:618–632., .
- 56Alzheimer's brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication. Proc Natl Acad Sci USA 2004; 101:10726–10731., , .
- 57Mitochondria in cybrids containing mtDNA from persons with mitochondriopathies. J Neurosci Res 2007; 85:3416–3428..
- 58Mitochondrial DNA mutations in disease and aging. J Cell Biol 2011; 193:809–818., .
- 59Aging and amyloid beta-induced oxidative DNA damage and mitochondrial dysfunction in Alzheimer's disease: implications for early intervention and therapeutics. Biochim Biophys Acta 2011; 1812:1359–1370., .
- 60Marked changes in mitochondrial DNA deletion levels in Alzheimer brains. Genomics 1994; 23:471–476., , et al.
- 61Mitochondrial DNA variants observed in Alzheimer disease and Parkinson disease patients. Genomics 1993; 17:171–184., , et al.
- 62Mitochondria dysfunction of Alzheimer's disease cybrids enhances Abeta toxicity. J Neurochem 2004; 89:1417–1426., , , .
- 63Alzheimer's disease cybrids replicate beta-amyloid abnormalities through cell death pathways. Ann Neurol 2000; 48:148–155., , et al.
- 64Endogenous oxidative stress in sporadic Alzheimer's disease neuronal cybrids reduces viability by increasing apoptosis through pro-death signaling pathways and is mimicked by oxidant exposure of control cybrids. Neurobiol Dis 2005; 19:312–322., , .
- 65Cybrids in Alzheimer's disease: a cellular model of the disease? Neurology 1997; 49:918–925., , et al.
- 66Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a. J Biol Chem 2000; 275:4177–4182., , , , .
- 67The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants. Hum Mol Genet 2004; 13:869–879., , et al.
- 68Melatonin as a natural ally against oxidative stress: a physicochemical examination. J Pineal Res 2011; 51:1–16., , .
- 69Extrapineal melatonin: analysis of its subcellular distribution and daily fluctuations. J Pineal Res 2012; 52:217–227., , et al.
- 70Critical role of mitochondrial reactive oxygen species formation in visible laser irradiation-induced apoptosis in rat brain astrocytes (RBA-1). J Biomed Sci 2002; 9:507–516., , , , .
- 71Mitochondrial reactive oxygen species generation and calcium increase induced by visible light in astrocytes. Ann N Y Acad Sci 2004; 1011:45–56., , , , .
- 72Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes. J Pineal Res 2004; 37:55–70., , , , , .
- 73Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55–63..
- 74In vivo measurements of respiration control by cytochrome c oxidase and in situ analysis of oxidative phosphorylation. Methods Cell Biol 2001; 65:119–131., .
- 75Calcium signaling and neurodegenerative diseases. Trends Mol Med 2009; 15:89–100..
- 76Calcium dysregulation in Alzheimer's disease. Neurochem Int 2008; 52:621–633., , .
- 77Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease. Nat Rev Neurosci 2002; 3:862–872..
- 78Neuronal and glial calcium signaling in Alzheimer's disease. Cell Calcium 2003; 34:385–397., .
- 79Calcium dysregulation in Alzheimer's disease: from mechanisms to therapeutic opportunities. Prog Neurobiol 2009; 89:240–255., , .
- 80Modulation of mitochondrial calcium as a pharmacological target for Alzheimer's disease. Ageing Res Rev 2010; 9:447–456., , .
- 81Beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci 1992; 12:376–389., , , , , .
- 82Fresh and nonfibrillar amyloid beta protein (1–40) induces rapid cellular degeneration in aged human fibroblasts: evidence for AbetaP-channel-mediated cellular toxicity. FASEB J 2000; 14:1244–1254., , .
- 83Calcium and mitochondria. FEBS Lett 2004; 567:96–102., , , , .
- 84Amyloid beta peptide 1–42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures. Cell Calcium 2012; 51:95–106., , , , , .
- 85Ca2+ storage capacity of rat brain mitochondria declines during the postnatal development without change in ROS production capacity. Antioxid Redox Signal 2007; 9:191–199., .
- 86Mitochondria and heart disease. Adv Exp Med Biol 2012; 942:249–267..
- 87Visualizing common deletion of mitochondrial DNA-augmented mitochondrial reactive oxygen species generation and apoptosis upon oxidative stress. Biochim Biophys Acta 2006; 1762:241–255., , et al.
- 88Mitochondrial fusion and fission in mammals. Annu Rev Cell Dev Biol 2006; 22:79–99..
- 89Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases. Hum Mol Genet 2009; 18:R169–R176., .
- 90Pathophysiological and pharmacological implications of mitochondria-targeted reactive oxygen species generation in astrocytes. Adv Drug Deliv Rev 2008; 60:1512–1526..
- 91Respiratory active mitochondrial supercomplexes. Mol Cell 2008; 32:529–539., , , , .
- 92The antioxidant N-acetylcysteine prevents the mitochondrial fragmentation induced by soluble amyloid-beta peptide oligomers. Neurodegener Dis 2012; 10:34–37., , , .
- 93Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 1995; 182:367–377., , et al.
- 94Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J Exp Med 2000; 192:1001–1014., , , , .
- 95Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta 2006; 1757:509–517., , .