Oligomeric and fibrillar species of β-amyloid (Aβ42) both impair mitochondrial function in P301L tau transgenic mice
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- Eckert, A., Hauptmann, S., Scherping, I. et al. J Mol Med (2008) 86: 1255. doi:10.1007/s00109-008-0391-6
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We recently provided evidence for a mitochondrial dysfunction in P301L tau transgenic mice, a strain modeling the tau pathology of Alzheimer’s disease (AD) and frontotemporal dementia (FTD). In addition to tau aggregates, the AD brain is further characterized by Aβ peptide-containing plaques. When we addressed the role of Aβ, this indicated a synergistic action of tau and Aβ pathology on the mitochondria. In the present study, we compared the toxicity of different Aβ42 conformations in light of recent studies suggesting that oligomeric rather than fibrillar Aβ might be the actual toxic species. Interestingly, both oligomeric and fibrillar, but not disaggregated (mainly monomeric) Aβ42 caused a decreased mitochondrial membrane potential in cortical brain cells obtained from FTD P301L tau transgenic mice. This was not observed with cerebellar preparations indicating selective vulnerability of cortical neurons. Furthermore, we found reductions in state 3 respiration, the respiratory control ratio, and uncoupled respiration when incubating P301L tau mitochondria either with oligomeric or fibrillar preparations of Aβ42. Finally, we found that aging specifically increased the sensitivity of mitochondria to oligomeric Aβ42 damage indicating that oligomeric and fibrillar Aβ42 are both toxic, but exert different degrees of toxicity.
KeywordsAlzheimer’s disease Amyloid aggregates Amyloid β-peptide Amyloid toxicity Fibrils Frontotemporal dementia Globulomer Mitochondria Oligomer Protein aggregation Respiration Tau Transgenic mice
β-Amyloid (Aβ)-containing plaques and tau-containing neurofibrillary tangles (NFTs) are hallmarks for brain lesions in both familial and sporadic cases of Alzheimer’s disease (AD); however, how these lesions and their proteinaceous components impair cellular functions and ultimately lead to neuronal cell loss is only partly understood [1, 2, 3]. Attempts to gain insight into pathogenic mechanisms, and eventually, to develop therapeutic strategies have been greatly assisted by experimental animal models that express mutant forms of AD-associated genes [4, 5].
In AD, pathogenic mutations have been identified in both the gene encoding the precursor of the Aβ peptide, APP, itself and in the PSEN genes which encode part of the APP–protease complex. No mutations have been identified in the MAPT gene encoding the microtubule-associated protein tau . Although AD is the most prevalent form of dementia at high age, NFTs are, in the absence of Aβ plaques, also abundant in additional neurodegenerative diseases, including frontotemporal dementia (FTD). In familial cases of FTD (FTD with Parkinsonism linked to chromosome 17 (FTDP-17)), exonic and intronic mutations have been identified in the MAPT gene which lead to the formation of tau aggregates in the brain . Gallyas silver impregnation techniques are frequently employed to visualize NFTs in both AD and FTD brains .
The P301L tau mutation was among the first to be identified in FTDP-17 . It was expressed by us in neurons of transgenic mice . P301L tau transgenic mice develop NFTs that contain aggregated forms of hyperphosphorylated tau and show age-related memory impairment [10, 11]. Whereas transgenic mice expressing human APP with mutations found in familial cases of AD develop Aβ plaques, they fail to form NFTs. On the other hand, the P301L transgenic pR5 mice model aspects of the tau pathology of AD and FTD, but lacks plaque pathology. Both pathologies have been successfully combined either by crossing the respective transgenic strains or by establishing so-called triple transgenic mice  (reviewed in ). We addressed the pathogenic relationship of Aβ plaques and NFTs by an alternative approach that is by injecting fibrillar preparations of Aβ42 stereotaxically into the somatosensory cortex and the hippocampus of P301L and wild-type human tau transgenic mice and non-transgenic littermate controls. As shown previously, this caused a fivefold increase of NFTs in the amygdala of P301L transgenic, but not wild-type tau transgenic or control mice . NFT formation was correlated with the phosphorylation of the pS422 and AT100 epitopes of tau [9, 15]. These findings could be confirmed by us in vitro, using neuronally differentiated SH-SY5Y neuroblastoma cells .
Subsequently, in addition to transcriptomic approaches [17, 18], we performed a proteomic and functional analysis of P301L tau transgenic mice which revealed a mitochondrial dysfunction together with a reduced electron transport chain complex I activity . We also provided first evidence for an increased vulnerability of P301L tau mitochondria towards fibrillar Aβ insult, suggesting a synergistic action of tau and Aβ pathology on the mitochondria [19, 20, 21].
In recent years, attempts have been undertaken to identify the toxic species of Aβ. The focus of attention has since shifted from fibrillar to oligomeric species as the large, insoluble Aβ deposits which form the amyloid plaques in the limbic and association cortices of AD patients are in equilibrium with small, diffusible Aβ oligomers that appear capable of interfering with hippocampal synaptic function and memory . Thus, in this study, we determined whether oligomeric Aβ42 would already exert a pronounced toxicity towards P301L tau transgenic mitochondria, compared to disaggregated (mainly monomeric) and fibrillar Aβ.
Materials and methods
The transgenic mice used in the present study express the human pathogenic mutation P301L of tau together with the longest human brain tau isoform (htau40) under control of the neuron-specific mThy1.2 promoter . The htau40 isoform contains exons 2 and 3 as well as four microtubule-binding repeats (2+3+4R, human tau40). Pronuclear injections were done into C57Bl/6 × DBA/2 F2 oocytes to obtain founder animals that were back-crossed with C57Bl/6 mice to establish transgenic lines as described . P301L tau mice show tau hyperphosphorylation already at 3 months . NFT formation starts at 6 months of age . Consistent with our previous studies , the mice were analyzed at 13–15 and 21–22 months of age. The mice were maintained in a 12-h dark–light cycle with pelleted food and water ad libitum. Animals were handled according to the Swiss guidelines for animal care. The experiments were conducted in accordance with international standards on animal welfare as well as being compliant with local regulations.
Monomer, oligomer, and fibril preparation
The Aβ42 synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) was suspended in 100% 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) at 6 mg/ml and incubated for complete solubilization under shaking at 37°C for 1.5 h as described previously .
Monomeric preparation 
To obtain the monomeric preparation, 30 mg HFIP-treated Aβ42 was resuspended in 132 μl dimethylsulfoxide (DMSO) at RT for 10 min. A 60 ml buffer (20 mM NaPi, 140 mM NaCl, and 0.1% Pluronic F68 pH 7.4) was added and the mixture stirred for 1 h at RT. Following a 20 min centrifugation at 3,000×g, the supernatant was discarded and the pellet resuspended in 6 ml buffer. Then, 34 ml of H2O was added and the mixture stirred for 1 h at RT. Following a second centrifugation for 20 min at 3,000×g, the supernatant was stored as 5 ml aliquots at −80°C.
Oligomeric preparation 
To obtain the oligomeric preparation, HFIP was removed by evaporation in a SpeedVac and Aβ42 resuspended at a concentration of 5 mM in DMSO and sonicated for 20 s as described . The HFIP-pretreated Aβ42 was diluted in phosphate-buffered saline (20 mM NaH2PO4, 140 mM NaCl, pH 7.4) to 400 μM and 1/10 volume 2% sodium dodecyl sulfate (SDS; in H2O) was added (final concentration of 0.2% SDS). An incubation for 6 h at 37°C resulted in the 16/20-kDa Aβ42 globulomer (short form for globular oligomer) intermediate. The 38/48-kDa Aβ42 globulomer was generated by a further dilution with three volumes of H2O and incubation for 18 h at 37°C. This was followed by centrifugation for 20 min at 3,000×g and concentrating the supernatant to 1.8 ml by dialysis against 5 mM NaPi, 35 mM NaCl pH 7.4 overnight at 6°C with a 30-kD centriprep and subsequent centrifugation of the concentrate for 10 min at 10,000×g. The supernatant was then stored in 100 μl aliquots at −80°C.
To prepare fibrillar Aβ42, the peptide was dissolved in Tris-buffered saline pH 7.4 (TBS) at a concentration of 0.5 mM and stored at −20°C. (All aqueous solutions were prepared with deionized and filtered water (Millipore)). The stock solution was diluted in TBS to a concentration of 50 μM and incubated at 37°C with gentle agitation for 24 h to obtain aged, aggregated preparations of Aβ42.
Visualization of Aβ preparations on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels
The samples were analyzed using Tris-glycine 4–20% gradient SDS-PAGE gels and stained with Coomassie Brilliant Blue R250 according to . Gels were scanned with an Epson 4180 scanner.
Structural characterization of Aβ aggregates
For negative staining analysis, 4 μl of the sample was placed on copper grids covered with formvar and carbon and counterstained with 2% uranyl acetate, using the droplet technique . Specimens were examined in a Zeus 902 transmission electron microscope operated at an acceleration voltage of 80 kV.
Thioflavine T (ThT) spectra were recorded at room temperature with a Shimadzu RF-5301PC fluorimeter, using an excitation wavelength of 482 nm and a cuvette with 5 mm path length. Samples contained a final concentration of 20 μM ThT and 5 μM Aβ42 in 5 mM NaPi/35 mM NaCl, pH 7.4 (globulomers) or TBS, pH 7.4 (fibrils).
Congo Red (CR) absorption spectra were recorded at room temperature, using an Analytik Jena Specord 210 spectrophotometer. Samples contained a final concentration of 10 μM CR and 15 μM Aβ42 in 5 mM NaPi/35 mM NaCl, pH 7.4 (globulomers) or TBS, pH 7.4 (fibrils).
For attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, Aβ42 fibrils were concentrated by centrifugation for 30 min at 500,000×g and 25°C in a Beckman Optima TLX ultracentrifuge, using a Beckman TLA-120.2 rotor. Subsequently, the fibril pellet was resuspended in a small volume of TBS, pH 7.4 by pipetting and vortexing. ATR-FTIR spectra of 1.9 mM Aβ42 globulomers in 5 mM NaPi/35 mM NaCl, pH 7.4 and 1.1 mM Aβ42 fibrils in TBS, pH 7.4 were recorded as described .
Brain tissue preparation for mitochondrial analysis
Cellular preparations were obtained to determine the mitochondrial membrane potential (MMP) and mitochondria and to determine respiration rates as previously described [19, 25]. For that, mice (six pairs of 13–15-month-old and six pairs of 21–22-month-old hemizygous P301L tau and WT control mice) were sacrificed by decapitation and brains quickly dissected on ice. The cerebellum and one cortical hemisphere (the other hemisphere was directly used for preparation of isolated mitochondria for mitochondrial respiration) were separately minced into 1 ml of medium I (138 mM NaCl, 5.4 mM KCl, 0.17 mM Na2HPO4, 0.22 mM K2PO4, 5.5 mM glucose, 58.4 mM sucrose, pH 7.35) with a scalpel and further dissociated by trituration through a nylon mesh (pore diameter 1 mm) with a pasteur pipette. The resulting suspension, which contained both neuronal (about 72%) and glial cells (about 26%), was filtered by gravity through a fresh nylon mesh with a pore diameter of 102 μm, and the dissociated cell aggregates were washed twice with medium II (110 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl2·H2O, 1 mM MgCl2·6 H2O, 25 mM glucose, 70 mM sucrose, 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 7.4) by centrifugation (400×g for 3 min at 4°C). One hundred microliters of the suspension were used for protein determination. After centrifugation, cells were resuspended in 3 ml DMEM, and then aliquots of 100 μl were distributed per well in a 48-well plate for measurement of the mitochondrial membrane potential. The preparations of cerebellar and cortical cells from P301L tau transgenic mice and WT littermate controls (cross-over design) were made within 2 h under the same conditions and in parallel and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Viability was found to be >90% using the MTT assay and trypan blue stain exclusion test. Data are expressed as fluorescence units per milligram protein.
Determination of the mitochondrial membrane potential
The membrane potential of the inner mitochondrial membrane was measured using the rhodamine 123 (Molecular Probes, Leiden, Netherlands) dye added to the cell culture medium at a final concentration of 0.4 μM for 15 min. Cells were washed twice with Hank’s balanced salt solution (Sigma, Germany), and fluorescence was determined with a Victor2 multiplate reader (Perkin Elmer, Rodgau-Jügesheim, Germany) at 490/535 nm (Ex/Em). Loading capacity of the dye within the membrane decreases when the mitochondrial membrane potential declines after damage, e.g., exposure to Aβ42. For the secondary insult with Aβ42, cells were incubated for 4 h with the different types of preparation as described above. For this purpose, monomeric and oligomeric preparations were handled with specific care to avoid destabilization of conformation. Thus, frozen aliquots were quickly thawed and immediately diluted to the final assay concentration (maximal incubation time at 37°C for 4 h only within the nanomolar concentration range ≤100 nM). Since pre-experiments had shown that a maximum effect of fibrillar Aβ42 with regard to a reduction of MMP was reached at a concentration of 50 nM whereas 100 nM did not further decrease MMP (data not shown), the latter concentration was omitted due to limited brain material.
Preparation of isolated mitochondria
Mice were sacrificed by decapitation and one brain hemisphere was rapidly dissected on ice and washed in ice-cold buffer (210 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM ethylenediamine tetraacetic acid (EDTA), 0.45% BSA, 0.5 mM DTT, protease inhibitor cocktail (Complete tablets, Roche Diagnostics). After removing the cerebellum and one cortical hemisphere for the determination of the membrane potential (see above), the second cortical hemisphere was homogenized in 2 ml buffer with a glass homogenizer (10 to 15 strokes, 400 rpm) followed by centrifugation at 1,400×g for 7 min at 4°C, to remove nuclei and tissue particles. This low speed centrifugation step was repeated once with the supernatant. Then, the supernatant fraction was centrifuged at 10,000×g for 5 min at 4°C to pellet mitochondria. The resulting pellet was resuspended in 1 ml ice-cold buffer and centrifuged again at 800×g for 3 min at 4°C. Finally, the mitochondria-enriched supernatant was centrifuged at 10,000×g for 5 min at 4°C to obtain a mitochondrial fraction. This fraction was resuspended in 100 μl of ice-cold buffer and stored at 4°C until use, followed by determination of protein content.
The rate of mitochondrial respiration was monitored at 25°C using an Oxygraph-2k system (Oroboros, Innsbruck, Austria) equipped with two chambers and DatLab software. Mitochondria (0.5 mg) were added to 2 ml of a buffer containing 65 mM sucrose, 10 mM potassium phosphate, 10 mM Tris–HCl, 10 mM MgSO4, and 2 mM EDTA (pH 7.0). State 4 respiration was measured after adding 40 μl malate/glutamate (240 mM/280 mM; assay concentration 4.8/5.6 mM). Then, 10 μl adenosine diphosphate (ADP; 100 mM; assay concentration 0.5 mM) was added to measure state 3 respiration. After determining coupled respiration, 1 μl carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.1 mM; assay concentration 0.05 nM) was added to the reaction chamber, and respiration measured in the absence of a proton gradient. To inhibit complex I activity, a total volume of 3 μl (2 + 1 μl) rotenone (0.1 mM; final concentration 0.15 nM) was added. Then, 10 μl succinate (1 M; final concentration 5 mM) was added and complex II-dependent respiration was measured. Finally, 8 μl KCN (0.5 M; assay concentration 2 mM) was added to inhibit complex IV activity. P301L tau transgenic and WT mitochondria were measured in parallel using the same conditions (cross-over design in the two chamber system). On a routine basis, the intactness of mitochondria was confirmed by addition of cytochrome c (10 μM). To test the effect of Aβ42 on mitochondrial respiration, isolated mitochondria were exposed to different types of Aβ42 preparations (5 μM) or vehicle on ice. Then, the mitochondria suspension was added into the chamber (final concentration of Aβ42 preparation 100 nM).
Data are represented as mean ± SEM. For statistical comparison, two-way ANOVA followed by post hoc t test was used. Only p values less than 0.05 were considered statistically significant.
We previously reported a mitochondrial dysfunction in P301L tau transgenic mice that is not caused by alterations in numbers of mitochondria . Furthermore, in aged P301L tau mice, we had found a significantly reduced state 3 respiration, and thus markedly reduced respiratory control ratio compared to age-matched WT mice. First evidence was provided that a secondary insult with Aβ42 caused a higher reduction in membrane potential in P301L tau mitochondria than in WT. Here, we aimed to determine the relative toxicity of oligomeric compared to fibrillar Aβ42 with regards to mitochondrial function, by testing disaggregated (mainly monomeric), oligomeric, and fibrillar preparations of Aβ42.
Characterization of Aβ42 species
The oligomeric preparation with an apparent molecular weight of 50 kDa was generated by a series of concentration, centrifugation, and dialysis steps. Figure 1b shows a Coomassie Blue staining of an SDS-PAGE gel after the initial preparation of Aβ42 (10 μl, lane 2), after concentration (0.5 μl, lane 3), after concentration and dialysis (0.5 μl, lane 4), and the flow through (10 μl, lane 5). After concentrating the sample (lanes 3 and 4), the preparation mainly consisted of oligomeric Aβ42, with monomeric Aβ42 and trimers and tetramers being only a minor contaminant. Negative staining electron microscopy revealed fibril-free preparations of spherical aggregates with a diameter of 2 to 5 nm (Fig. 1c). Aβ42 globulomers bind weaker to the fluorescent amyloid-specific dye thioflavine T (ThT) compared to mature fibrils, as indicated by the smaller fluorescence intensity signal in the ThT spectrum (Fig. 1e,f). Moreover, these aggregate species do not exhibit an increased optical absorption when stained with Congo Red (Fig. 1g). The FTIR spectrum of the Aβ42 globulomers provides further information on their secondary structure (Fig. 1i). The maximum of their amide I′ band is at 1,628 cm−1 and, therefore, within the range that is typically observed for β-sheet-rich amyloid fibrils . However, the FTIR spectrum of the globulomers shows a clearly resolvable peak at 1,693 cm−1, which has also been observed for Aβ40 oligomeric aggregate species and might indicate antiparallel β-sheet structure . For the globulomers, the contribution of the main peak at 1,628 cm−1 to the amide I′ band is smaller than for Aβ42 fibrils, suggesting that they contain less β-sheet structure.
Natural human Aβ oligomers are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell . Previous studies have shown that these Aβ42 globulomers, as they have been also termed, are a persistent structural entity formed independently of the fibrillar aggregation pathway . These preparations are a potent antigen in mice and rabbits eliciting the generation of Aβ42 globulomer-specific antibodies that do not cross-react with the full-length amyloid precursor protein APP, Aβ40, and Aβ42 monomers or Aβ fibrils. Furthermore, Aβ42 globulomers have been shown to bind specifically to dendritic processes of neurons but not glia in primary hippocampal cultures and to completely block long-term potentiation (LTP) in rat hippocampal slices .
Fibrillar Aβ42 was obtained by resuspension of Aβ42 in TBS and an incubation at 37°C for 24 h as described [16, 25]. Negative staining electron microscopy clearly shows mature, twisted Aβ42 fibrils with a width of 6 to 12 nm (Fig. 1d). Aβ42 fibrils show amyloid-typical tinctorial properties upon binding to ThT and CR (Fig. 1f,h). The fluorescence intensity of ThT at 482 nm and the CR absorption increase substantially in the presence of Aβ42 fibrils. Moreover, the maximum of the CR absorption is shifted from 497 to 513 nm, as typically observed for amyloid fibrils . The amide I′ band of the fibrils also has its maximum at 1,628 cm−1 and suggests the presence of an amyloid-like extended β-sheet structure in Aβ42 fibrils (Fig. 1j). Within the limits of detection, this preparation was free of oligomers (data not shown).
Both oligomeric and fibrillar Aβ42 cause a decreased membrane potential in P301L tau cortical preparations
We dissected the cerebellum and two cerebral brain hemispheres from 6 P301L transgenic and six non-transgenic control WT littermates at 13–15 months of age. The cerebellum and one cerebral hemisphere were triturated separately to obtain cellular preparations to determine the mitochondrial membrane potential. The second cerebral hemisphere was used to prepare isolated mitochondria to determine the rate of mitochondrial respiration. Brain cells and isolated mitochondria from the same animals were treated in parallel with the different Aβ42 preparations.
We conclude from this that cortical preparations of both WT and P301L tau transgenic mice are susceptible to Aβ42 preparations, be they oligomeric or fibrillar in nature. Importantly, when we looked at cerebellar preparations, they were resistant to all three types of Aβ42 preparations in as much as they showed no impairment of the MMP (Fig. 2d). This finding underscores the concept of selective vulnerability in neurodegeneration. For example, for the Aβ-associated pathology in AD, five phases have been defined, with only the final fifth phase being characterized by Aβ deposition in the cerebellum [31, 32]. Similarly, NFTs are found in the cerebellum only in the final stages of AD, and in FTD, they are also not frequently encountered . The mechanisms underlying selective vulnerability in AD and FTD are largely unknown, and several hypotheses have been put forward. Interestingly, one of the factors determining which cells die first when different types of cells are exposed to the same stress may be variations in mitochondrial composition .
Reduced state 3 respiration, respiratory control ratio, and uncoupled respiration in P301L tau mice
We have previously shown a significantly reduced state 3, but not state 4, respiration in aged (24-month-old) P301L tau transgenic compared to age-matched WT mice; this led to a markedly reduced respiratory control ratio .
We found in addition, after uncoupling with FCCP, that the respiratory rate in the absence of a proton gradient, i.e., the ‘uncoupled respiration’, was significantly diminished in 13–15-month-old P301L tau mice after incubation with either oligomeric or fibrillar preparations of Aβ42 (Figs. 3b and 4d) indicating a reduced maximum capacity of the electron transport chain. After complete inhibition of complex I with rotenone, succinate was added as a substrate for complex II. When the succinate-dependent respiration was normalized to the uncoupled complex I-dependent respiration in the respective experiments, there was no significant difference between treated and untreated P301L tau transgenic mitochondria (Fig. 3b). This indicates that neither complex II nor complex III and IV are decisively impaired by Aβ42. In accordance with the respiratory control ratio, we have previously shown that ATP levels of cerebral cells were unchanged in 12-month-old P301L tau transgenic mice, but significantly reduced with aging . Previous studies using homogenates of fresh samples of frontal neocortex from patients with dementia and neurosurgical controls suggested partial mitochondrial uncoupling in disease as the ratio of oxygen uptake rates in the presence and absence of ADP was significantly reduced for the dementia patients compared with controls . These in vitro results indicate that metabolic changes may be relevant to the pathogenesis of AD and related dementias .
Together, our results suggest that P301L tau mice exhibit an initial defect in mitochondrial function with reduced complex I activity, which is exacerbated by the presence of either oligomeric or fibrillar Aβ42.
Aging increases the sensitivity of mitochondria to oligomeric Aβ damage
Mitochondrial respiratory chain failure has been implicated as a factor in general aging and is likely to have a greater effect in tissues with a high dependency on energy generated through oxidative phosphorylation such as the brain . We have previously shown that aging contributes to the mitochondrial dysfunction in P301L tau mice . Furthermore, we had found a 62.3% reduction of complex V levels in human FTDP-17 brains obtained from carriers of the P301L mutation, compared to control brains . The decreased levels of complex V in human P301L FTDP-17 brains confirmed our proteomics observation made in the P301L tau transgenic mice and suggested that the P301L mutant tau pathology causes potentially a specific mitochondrial dysfunction in humans as well as in mice .
Together, aging increased sensitivity of cortical mitochondria to Aβ damage. This is in agreement with studies in APP transgenic mice  as well as in primates, where complex I activity had been shown to be reduced with aging . Thus, aging potentially intensifies complex I activity defects in the P301L tau mice.
Our findings first of all support the notion of a toxic role of Aβ in respiration and, together with our previous findings, antioxidant defense mechanisms [19, 25, 39]. Secondly, they reveal a role for tau in mitochondrial dysfunction. Thirdly, our experiments extend our previous findings that treatment of PC12 cells with extracellular Aβ causes a significant decrease in mitochondrial membrane potential . Finally, our results in P301L tau mice indicate the effects of both fibrillar and oligomeric Aβ on mitochondrial function of cortical brain cells at very low and physiological relevant concentrations within the nanomolar range and within a very short time frame (incubation time 4 h) indicating early and chronic alterations of mitochondrial functions that may impact overall neuronal homeostasis. In a related study, acute treatment of human cortical neurons with oligomeric Aβ (5 μM) for only short periods was shown to be sufficient to activate a mitochondrial apoptotic death pathway .
How does Aβ exert its effect on mitochondria? Interaction of Aβ with Aβ binding alcohol dehydrogenase (ABAD), a short-chain alcohol dehydrogenase in the mitochondrial matrix, has been shown to lead to mitochondria failure, e.g., mitochondrial membrane permeability and reduction of the activities of enzymes involved in mitochondrial respiration . ABAD also binds to oligomeric Aβ42 that has been found in cortical mitochondria of APP transgenic mice . Protease sensitivity assays suggest that Aβ gains access to the mitochondrial matrix rather than simply being adsorbed to the external surface of mitochondria . This may explain how Aβ42 affects mitochondrial membrane potential and respiration. In our studies, however, we did not determine whether there is a direct interaction between Aβ and mitochondria, nor whether extracellular Aβ is taken up by the neurons. It is very likely that within the short time frame of our experiments (i.e., 4 h) very little Aβ42 is taken up at all. The effects of this putatively minor fraction cannot be discriminated from that elicited by the majority of Aβ that is not taken up. Therefore, our experimental design does not allow drawing any conclusions concerning the role of intracellular Aβ in mitochondrial dysfunction. In any case, it is generally not understood how Aβ exerts its toxicity in other experimental paradigms, whether it is receptor mediated and if so whether it requires uptake of Aβ by nerve terminals and retrograde transport, whether it is dependent on pore formation and calcium ion influx, or whether it is related to damage to nerve terminals, by interacting with the lipid bilayer . It has been hypothesized that oligomeric Aβ, with its sharp morphology in contrast to monomeric Aβ, has the ability to permeabilize cellular membranes and lipid bilayers thereby entering organelles, such as the mitochondria [46, 47]. Of note, early reports about the action of aggregated Aβ on membranes implicate increased membrane permeability elicited by fibrils [48, 49]. These mechanisms might explain why aggregated Aβ preparations elicit effects on mitochondrial function, but not disaggregated Aβ.
The differences we found between the effects of fibrillar and oligomeric Aβ are subtle. In a related study, their effects have been dissected from monomers. Cell medium containing oligomers and abundant Aβ monomers, but not amyloid fibrils, were microinjected into rat brain and shown to markedly inhibit hippocampal long-term potentiation . Immunodepletion from the medium of all Aβ species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Aβ monomers but not oligomers, did not prevent the inhibition of LTP, indicating a role for Aβ oligomers. These were shown to disrupt synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid, in the absence of monomeric or fibrillar amyloid. When cells were treated with γ-secretase inhibitors at doses which prevented oligomer formation but allowed appreciable monomer production, this no longer disrupted LTP, indicating that synaptotoxic Aβ oligomers can be targeted therapeutically [30, 50]. In Neuro-2A cells, oligomers were shown to induce a tenfold greater increase in neurotoxicity as compared to fibrils . However, whereas LTP seems to be inhibited by oligomeric Aβ only and not by fibrillar Aβ, in a different experimental paradigm, as in our study, the two species seem to have both toxic, yet diverse effects . Using rat astrocyte cultures, oligomeric Aβ42 was shown to induce initial high levels of the pro-inflammatory molecule IL-1β that decreased over time, whereas fibrillar Aβ caused increased levels over time . Oligomeric, but not fibrillar Aβ, induced high levels of iNOS, NO, and TNFα, suggesting that oligomers induce a profound, early inflammatory response, whereas fibrillar Aβ shows less increases of pro-inflammatory molecules, consistent with a more chronic form of inflammation . Similarly, in our experimental paradigm, we found toxic effects of both fibrillar and oligomeric Aβ, but those of oligomeric Aβ were more pronounced.
The presence of the pathogenic P301L mutation of tau severely increased the susceptibility of mitochondria to Aβ preparations. How tau accumulation is mediating these changes is unclear. Overexpression of wild-type tau in cell culture has been shown to impair plus-end-directed axonal transport resulting in a reduction of mitochondria . However, this is unlikely the case for P301L tau mice, as mitochondrial neuritic numbers counted proximal or distal to the cell body, did not vary significantly compared to numbers in WT mice, . Also, the total amount of mitochondria has been shown to be unaltered in the transgenic mice . This is consistent with the finding of similar numbers of mitochondria between NFT and non-NFT-bearing cells in AD . It is likely that tau accumulation has direct effects on the mitochondria as the accumulation of increasingly insoluble ATP synthase α-chain together with NFTs has been shown in AD brains while detergent-soluble levels were reduced .
The synergistic effects of Aβ42 on mitochondria isolated from P301L tau transgenic mice may seem subtle, irrespective of whether they are incubated with fibrillar or oligomeric Aβ42, but these effects are of in vivo relevance as AD is a chronic and slowly progressive disease, with time spans of up to several decades between Aβ and tau aggregation and the onset of clinical symptoms.
In conclusion, we found that both fibrillar and oligomeric Aβ42 preparations impaired mitochondrial membrane potential and respiration. Furthermore, we revealed the synergistic effects of Aβ42 and P301L tau (see also: ). However, as aged cortical brain mitochondria showed an increased sensitivity to oligomeric compared to fibrillar Aβ, this suggests that oligomeric Aβ may be particularly toxic. Although it is likely that oligomeric Aβ represents the primary toxic insult, further experiments are needed to substantiate this notion. Thus, there is increasing evidence for different modes of toxic actions of Aβ suggesting that in the development of treatment strategies, targeting either oligomeric or fibrillar Aβ may not be sufficient, but the best approach is likely one that either prevents formation of excess Aβ altogether or assists in its rapid clearance.
This research was supported in part by grants from the SNF (Swiss National Science Foundation) #310000-108223 and Eli Lilly International Foundation to AE, and from the NHMRC, the ARC, the New South Wales Government through the Ministry for Science and Medical Research (BioFirst Program), the Medical Foundation (University of Sydney), and the Judith Jane Mason & Harold Stannett Williams Memorial Foundation to JG. JG is a Medical Foundation Fellow. We are grateful to Abbott GmbH & Co KG, Ludwigshafen, Germany, for the kind gift of monomeric and oligomeric Aβ42 preparations. M.F. is supported by grants from BMBF (BioFuture) and DFG.
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