Protection against oxidative stress in vitro
Beside the use of H2O2 in some initial experiments, we usually induced oxidative stress by the NO donor sodium nitroprusside (SNP), due to the important role of nitrosative stress in AD (Keil et al., 2004a,b). In PC12 cells, SNP, led to a reduction of MMP and ATP levels. Under basal conditions without additional SNP damage, piracetam did not affect both alterations even at rather high concentrations. However, piracetam was able to reduce both measures of mitochondrial dysfunction after pre- and post-incubation (Keil et al., 2006).
Another stressor, serum deprivation also leads to a decrease of mitochondrial membrane potential (MMP) and a decrease of ATP levels in neurons. Additionally, serum like glucose deprivation in PC12 cells causes peroxidation of their cell membrane lipids, decreases intracellular SOD activity, and enhances apoptosis in PC12 cells (Keil et al., 2004b, 2006). Interestingly, piracetam was able to protect MMP against cellular stress following serum deprivation. Under conditions of mild serum deprivation, when serum concentrations not lower than 2% were used, piracetam (500 μM) induced a nearly complete recovery of MMP. Furthermore, reduced ATP levels were already seen at 10% serum. Under this condition, piracetam completely restored ATP levels, while at lower serum concentrations only a partial restoration was seen (Keil et al., 2006).
As mentioned before, complex I and complex IV functions are impaired in aging and AD. Thus, the possible efficacy of piracetam to protect individual complexes of the mitochondrial respiratory chain after treatment with specific complex inhibitors was also investigated. Complexes I, II, and III were already protected at concentrations as low as 500 μM piracetam, while a significant protection of complexes IV and V was observed at a concentration of 1000 μM piracetam (Keil et al., 2006). This broad activity of piracetam is in line with the assumptions that improvement of complex activity might be due rather to its fluidity enhancing properties at mitochondrial membranes rather than by specific effects at the individual complexes. This is also supported by the data of Zhang et al. (2010) indicating enhanced activity of complexes I–IV in mice after induction of mitochondrial dysfunction with d-galactose. It is very important to note that the beneficial effects of piracetam have not only been seen at the level of MMP but also in using several other measures of mitochondrial function since MMP alterations are not always directly connected with changes of mitochondrial function (Cao et al., 2007; Kahlert et al., 2008).
As already mentioned, a major link between the mitochondrial defects of our brain accumulating during decades of aging and the specific Aß related toxicity in AD seems to be oxidative stress induced by Aß as well as Aß induced impairment of mitochondrial function, e.g., reduced activity of the complexes I and IV of the respiratory chain. This very slow process can experimentally be investigated by inducing mitochondrial dysfunction in cells in tissue culture following incubation with extracellularly applied Aß (Kurz et al., 2010). Since Aß1–42 is presently considered as the main toxic Aß species, we used this peptide and several experimental cell models (PC12 cells, HEK cells, dissociated mouse brain cells) to study possible protective effects of piracetam on mitochondrial deficits. When PC12 cells were treated with fibrillar Aß1–42 10 nM for 24 h a reduction of MMP was observed as described previously by our group (Keil et al., 2004a). The addition of piracetam 30 min after Aß1–42 substantially protected MMP at concentrations already beginning with 0.1 mM. Comparable protective effects of piracetam were observed for dissociated brain cells of Naval Medical Research Institute (NMRI) mice following incubation with fibrillar Aß1–42. In both cell types, piracetam alone has no effect on MMP.