Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid ␤ (A␤) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial A␤ levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular A␤ production by a functional ␥-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of A␤ in these processes. Extracellular treatment of PC12 cells with comparable A␤ concentrations only leads to weak changes, demonstrating the important role of intracellular A␤. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable A␤ levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased A␤ levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death.

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Evidence suggests that amyloid-beta (Ab) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Ab can lead to neuronal dysfunction. Here we investigated the specific effects of Ab on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Ab protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demise.

Autosomal dominant forms of familial Alzheimer's disease (FAD) are caused by mutations of the amyloid precursor protein (APP) gene and by mutations of the genes encoding for presenilin 1 or presenilin 2. Simultaneously, evidence is provided that increased oxidative stress might play a crucial role in the rapid progression of the Swedish FAD. Here we investigated the effect of the Swedish double mutation (K670M/N671L) in the ␤-amyloid precursor protein on oxidative stress-induced cell death mechanisms in PC12 cells. Western blot analysis and cleavage studies of caspase substrates revealed an elevated activity of the executor caspase 3 after treatment with hydrogen peroxide in cells containing the Swedish APP mutation. This elevated activity is the result of the enhanced activation of both intrinsic and extrinsic apoptosis pathways, including activation of caspase 2 and caspase 8. Furthermore, we observed an enhanced activation of JNK pathway and an attenuation of apoptosis by SP600125, a JNK inhibitor, through protection of mitochondrial dysfunction and reduction of caspase 9 activity. Our findings provide evidence that the massive neurodegeneration in early age of FAD patients could be a result of an increased vulnerability of neurons through activation of different apoptotic pathways as a consequence of elevated levels of oxidative stress.

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The presumption to suffer from Alzheimer's disease (AD) accelerates with aging. One important risk factor seems to be the isoform epsilon 4 of the apolipoprotein E gene (Apo epsilon 4), which increases the risk to develop AD at an earlier age. Furthermore, convincing evidence is provided that apoptotic cell death mechanisms play an important role in neuronal cell death in AD. In the present study, we investigated whether abnormalities in apoptosis and caspase-3 activity can be found at the level of lymphocytes and a T cell subtype, CD4 T cells, from AD patients compared to aged sex- and ApoE genotype-matched non-demented controls. Under different experimental conditions (at baseline or after in vitro incubation in the presence of proapoptotic stimuli) increased levels of apoptosis and enhanced caspase-3 activity were detected in lymphocytes from AD patients. This difference was most pronounced in the CD4(+) T cell subtype. Notably, we found a significant increase of apoptotic cells and caspase-3 activity in lymphocytes from AD patients bearing one or two alleles of the ApoE4 compared to non-E4 carriers. Again, these effects were strongest in CD4(+) T cells. Circulating amyloid-beta (A beta) levels did not differ between AD patients bearing ApoE4 and non-ApoE4 and age-matched controls. Therefore, it is likely that circulating A beta is not responsible for the observed effects, which might rather reflect an ongoing systemic response in AD, e.g. an increase in CD95 expression.

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