Protection against oxidative stress in dissociated brain cells from subchronically treated mice
After demonstrating piracetam's efficacy in reducing mitochondrial dysfunction induced by several stressors in vitro, we also investigated its protective effects following treatment of aged mice or mice transgenic for the Swedish human Amyloid Precursor Protein (APP) gene which express a substantial amount of Aß in the brains (tgAPP mice) (Keil et al., 2006; Kurz et al., 2010). The rational for using aged animals mainly originates from a large number of findings that piracetam's efficacy to improve cognitive functions usually is less pronounced in young healthy animals but gets much more prominent in aged animals (see Chapter 1.0). We used aged NMRI mice which are bread in our animal facilities not only showing clear cognitive impairment but also several measures of oxidative stress, and mitochondrial dysfunction at an age around 20 months including a decreased MMP compared to young mice (Müller et al., 2010). Piracetam treatment for 14 days normalized MMP in aged mice, while similar treatment of young animals had no effect (Figure 1).
On the other hand, a similar piracetam treatment not only protected brain cells of aged but also young mice against oxidative stress induced in vitro by addition of H2O2, SNP, or Aß1–42. Even if brain cells of young mice treated with piracetam also showed some benefit from piracetam treatment, aged animals usually responded most (Figure 1).
Antioxidative enzymes are the primary defense mechanism to protect biological macromolecules from oxidative damage, and are upregulated in aged mouse brain as an adaptive response to oxidative stress. Therefore, we investigated the effect of piracetam treatment on the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) in young mice (2–3 months old) and old mice (22–24 months old). We confirmed a significant increase in GPx and GR activity in aged mice compared to young mice. The activity of SOD had also a tendency to increase with age. Piracetam treatment decreased the activities of all three enzymes in aged mice nearly to the level of young animals. In young mice, a only small and not significant decrease of antioxidative enzymes could be observed (Figure 1). Quite similar mitochondria protecting effects have recently been observed in mice following experimentally induced mitochondrial dysfunction (g-galactose) after treatment with piracetam (300 mg/kg, 14 days) at the levels of MMP, activities of complexes I–IV, and ROS generation (Zhang et al., 2010). Unfortunately, both studies did not report glutathione levels, which also has a relevant role in regulating mitochondrial function (Jha et al., 2000).
Isolated brain cells of mice overexpressing mutated human amyloid precursor protein (tgAPP) show significant reductions of MMP and ATP synthesis relative to non-transgenic littermate controls confirming previous observation from our group (Hauptmann et al., 2009). Similar piracetam treatment (0.5 g/kg/day orally) already described for NMRI mice above again showed substantial improvement of MMP and ATP production (Figure 2). As reported earlier (Blanchard et al., 2003), these mice express substantial level of soluble Aß in the brain while littermates do not. Quite interestingly, piracetam treatment led to an about 25% reduction of soluble Aß (Kurz et al., 2010) (Figure 3). A related observation showing reduced Aß levels in the plasma of geriatric patients treated with piracetam was published by Blasko et al. (2005). In order to investigate if this effect of piracetam on Aß levels might also be associated with improved mitochondrial function, we used APPwt HEK 293 cells stably overexpressing human APP showing moderately enhanced Aß levels (Keil et al., 2004a). Piracetam lowered Aß levels under basal conditions (Kurz et al., 2010). In agreement with other findings (Guglielmotto et al., 2009) mitochondrial dysfunction induced with SNP elevates Aß1–40 levels substantially. Again, treatment with piracetam lowered Aß significantly by 15–20% (Figure 3). In addition, piracetam improves mitochondrial function under the same conditions in APPwt HEK 293 when Aß generation is decreased (Figure 3).
Figure 2  Piracetam improves measures of mitochondrial function in tgAPP mice. (A) Animals were treated for 14 days with 0.5 g piracetam/kg in 0.9% NaCI solution p.o. once daily for 2 weeks. Control animals received 0.9% NaCI solution alone. All data are modified after Kurz et al. (2010). The MMP was significantly reduced in tgAPP mice. Piracetam treatment normalizes the MMP to non-tgAPP levels. Data are expressed as mean ± SEM (n = 7–8). +p < 0.05 control non-tgAPP vs. control tgAPP, *p < 0.01 piracetam treated tgAPP vs. tgAPP control; Student's unpaired t-test. (B) ATP levels were also significantly impaired in tgAPP mice. In contrast, piracetam treatment increases ATP levels not only in tgAPP animals but also in control animals. Data are expressed as mean ± SEM (n = 7-8). *p < 0.05 control non-tgAPP vs. piracetam treated non-tgAPP; +p < 0.05 control non-tgAPP vs. control tgAPP, *p < 0.01 piracetam treated tgAPP vs. tgAPP control; Student's unpaired t-test.
Figure 3  Piracetam ameliorates elevated Aβ production following mitochondrial dysfunction. (A) Normalized Aβ levels were quantified in brain homogenates from non-tg littermate and tgAPP mice (3 months old). **p < 0.01 piracetam treated tgAPP vs. control tgAPP; student's unpaired t-test. (B) Piracetam reduces nitrosative stress induced elevation of Aβ in APPwt HEK293 cells. Cells were preincubated for 24 h with piracetam (1 mM) and stressed for additional 24 h with SNP (0.5 mM). Data are expressed as mean ± SEM (n = 3–4). +++p < 0.001 APPwt HEK293 control cells vs. cells treated with SNP, *p < 0.01 APPwt HEK293 cells stressed with SNP 0.5 mM vs. stressed cells preincubated with piracetam; Student's unpaired t-test. (C) Piracetam ameliorates nitrosative stress induced reduction of mitochondrial membrane potential. Cells were preincubated for 24 h with piracetam (1 mM) and stressed for additional 24 h with SNP (0.5 mM). Data are expressed as mean ± SEM (n = 6). All data are modified after Kurz et al. (2010). **p < 0.01 APPwt HEK293 cells stressed with SNP 0.5 mM vs. stressed cells preincubated with piracetam, Student's unpaired t-test.