Table 1.
Primers used in this study.
Figure 1.
Treatment of HtrA2 KO animals with Idebenone and Reseveratrol.
A Both Idebenone (orange line, 25 animals) and Reseveratrol (green line, 34 animals) significantly increased the mean survival time of HtrA2 KO mice compared to sham-treated animals (black line, 77 animals,). In addition, Idebenone increased the maximal life span. B Grid test of HtrA2 KO animals. Both compounds extend the period of life during which the KO animals can perform the motor task successfully (Idebenone: 10 animals, resveratrol: 6 animals, untreated 5 animals).
Figure 2.
Quantification of neuronal density in dorsal part of striatum.
A NeuN staining in the dorsal part of the striatum. Scale bar: 280 µm. B The treatment with Idebenone and Reseveratrol could significantly attenuate neuronal loss in the dorsal part of the striatum of KO mice. Six mice/treatment group were analyzed.
Figure 3.
Relative expression of different genes involved in the pathogenesis of HtrA2 KO.
A Relative expression of CHOP in the striatum. HtrA2 KO animals showed elevated levels of CHOP levels compared to wt. After treatment with Idebenone HtrA2 KO animals showed reduced levels of CHOP. B Relative expression of ATF4 in the striatum. Levels of ATF4 were elevated in HtrA2 KO mice compared to wt, but not influenced by Idebenone or Reseveratrol treatment. C Relative expression of Bcl2 in the striatum. The relative expression of Bcl2 was not up-regulated in HtrA2 KO animals. Neither treatment with Idebenone nor with Reseveratrol changed the relative expression of Bcl2. D Relative expression of Bax in the striatum. HtrA2 KO animals exhibited elevated expression levels of Bax. Treatment with Reseveratrol attenuated the up-regulation of Bax. E Relative expression of Noxa in the striatum. HtrA2 KO animals showed increased relative expression of Noxa. Treatment with Idebenone of HtrA2 KO animals induced down-regulation of Noxa. F Relative expression of p53 in the striatum. HtrA2 KO animals showed upregulated expression levels of p53, while treatment with Idebenone and Reseveratrol had no effect on this. G Relative expression of DR5 in the striatum. The elevated levels of DR5 could be reduced after treatment with Idebenone. Empty bars: wt, grey bars: HtrA2 KO. Data are mean± SD; *** p<0.001, ** p<0.01, * p<0.05, ns: not significant; n = 6 mice/group.
Figure 4.
Signaling cascades involving CHOP in the striatum of HtrA2 KO mice.
The existence of HtrA2 appears to be crucial for disease progression characterized by mitochondrial dysfunction, which contributes to neuronal cell death via up-regulation of the transcription factors ATF4 and CHOP. CHOP is known to repress Bcl2 (pro-survival) gene expression, which increases the proportion of pro-apoptotic Bcl2 proteins such as Bax. Oxidative stress also leads to an up-regulation of the pro-apoptotic Bcl2- protein Noxa, which can be activated by the transcription factor p53. Noxa exerts its pro-apoptotic function by neutralizing the pro-survival Bcl2 protein Mcl1/Al, thereby facilitating the activation of Bax/Bak proteins. CHOP is also known to regulate the extrinstic apoptotic signaling pathway by activating Death Receptor 5 (DR5), a member of the TNFR superfamily, by binding to its promoter region. Idebenone excerts anti-apoptotic effects on CHOP- and Noxa activation downstream of ATF4 and p53, respectively. Idebenone induces also anti-apoptotic effects on DR5 expression. Reseveratrol has been shown to reduce the expression of Bax, downstream of the CHOP- and Noxa activation cascades. Pro-apoptotic factors placed in grey boxes and anti-apoptotic factors placed in white boxes. Antioxidant compounds, used in this study, placed in white boxes with double lines, dashed lines stay for dependent interactions known from literature, solid lines represent dependent interactions drawing from experimental data shown her.