Abstract
Impaired mitochondrial integrity and function are key features of intrinsic death pathways in neuronal cells. Therefore, key regulators of intrinsic death pathways acting upstream of mitochondria are potential targets for therapeutic approaches of neuroprotection. The tumor suppressor p53 is a well-established regulator of cellular responses towards different kinds of lethal stress, including oxidative stress. Recent reports suggested that p53 may affect mitochondrial integrity and function through both, transcriptional activation of mitochondria-targeted pro-death proteins and direct effects at the mitochondrial membrane. In the present study, we compared the effects of pharmacological inhibition of p53 by pifithrin-α with those of selective p53 gene silencing by RNA interference. Using MTT assay and real-time cell impedance measurements we confirmed the protective effect of both strategies against glutamate-induced oxidative stress in immortalized mouse hippocampal HT-22 neurons. Further, we observed full restoration of mitochondrial membrane potential and inhibition of glutamate-induced mitochondrial fragmentation by pifithrin-α which was, in contrast, not achieved by p53 gene silencing. Downregulation of p53 by siRNA decreased p53 transcriptional activity and reduced expression levels of p21 mRNA, while pifithrin-α did not affect these endpoints. These results suggest a neuroprotective effect of pifithrin-α which occurred at the level of mitochondria and independently of p53 inhibition.
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Abbreviations
- AIF:
-
Apoptosis inducing factor
- AD:
-
Alzheimer’s disease
- ANOVA:
-
Analysis of variance
- Bid:
-
BH3 interacting-domain death agonist
- CCCP:
-
Carbonyl cyanide 3-chlorophenylhydrazone
- DRP1:
-
Dynamin-related protein 1
- FACS:
-
Fluorescence-activated cell sorting
- GAPDH:
-
Glyceraldehyde 3-phosphate dehydrogenase
- LPS:
-
Lipopolysaccharide
- MDM2:
-
Mouse double minute 2 homolog
- MMP:
-
Mitochondrial membrane potential
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PCD:
-
Programmed cell death
- PD:
-
Parkinson’s disease
- PFTα:
-
Pifithrin-α
- pMCAO:
-
Permanent middle cerebral artery occlusion
- PUMA:
-
p53 upregulated modulator of apoptosis
- ROS:
-
Reactive oxygen species
- TIF-IA:
-
Pol I-specific transcription initiation factor IA
- TMRE:
-
Tetramethylrhodamin ethyl ester
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Acknowledgments
We thank the excellent technical support by Mrs. Katharina Elsässer and Eileen Daube. Moreover, we thank Mrs. Emma Esser for careful editing of the manuscript and Roche Diagnostics GmbH for providing support with the xCELLigence system.
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The authors declare that they have no conflict of interests.
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Neitemeier, S., Ganjam, G.K., Diemert, S. et al. Pifithrin-α provides neuroprotective effects at the level of mitochondria independently of p53 inhibition. Apoptosis 19, 1665–1677 (2014). https://doi.org/10.1007/s10495-014-1048-2
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DOI: https://doi.org/10.1007/s10495-014-1048-2