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Published on: April 10, 2013
Proteome analysis of DNA damage-induced neuronal death using high throughput mass spectrometry
Mark D Johnson1, Li-Rong Yu, Thomas P Conrads
1Department of Neurological Surgery, University of Washington School of Medicine, Seattle, Washington 98195-6470, USA.
Abstract:
Isotope-coded affinity tag reagents and high throughput mass spectrometry were used to quantitate changes in the expression of 150 proteins in mouse wild-type (p53(+/+)) cortical neurons undergoing DNA damage-induced death. Immunological techniques confirmed several of the changes in protein expression, but microarray analysis indicated that many of these changes were not accompanied by altered mRNA expression. Proteome analysis revealed perturbations in mitochondrial function, free radical production, and neuritogenesis that were not observed in p53-deficient neurons. Changes in Tau, cofilin, and other proteins recapitulated abnormalities observed in neurodegenerative states in vivo. Additionally, DNA damage caused a p53-dependent decrease in expression of members of the protein kinase A (PKA) signaling pathway. PKA inhibition promoted death in the absence of DNA damage, revealing a novel mechanism by which endogenous down-regulation of PKA signaling may contribute to p53-dependent neuronal death. These data demonstrate the power of high throughput mass spectrometry for quantitative analysis of the neuronal proteome.
Insights
High-throughput mass spectrometry revealed p53-dependent protein changes in neurons during DNA damage-induced death. These alterations, impacting mitochondrial function and PKA signaling, mimic neurodegenerative states and suggest novel death mechanisms.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- DNA damage triggers neuronal death, a process implicated in neurodegenerative diseases.
- The role of the p53 protein in neuronal apoptosis and its downstream molecular targets remain incompletely understood.
Purpose of the Study:
- To quantitatively analyze proteome-wide changes in mouse cortical neurons undergoing DNA damage-induced death.
- To investigate the involvement of the p53 protein in these proteomic alterations.
- To identify novel molecular pathways contributing to p53-dependent neuronal death.
Main Methods:
- Quantitative proteomic analysis using isotope-coded affinity tag (ICAT) reagents and high-throughput mass spectrometry.
- Confirmation of protein expression changes using immunological techniques.
- Microarray analysis to compare mRNA and protein expression levels.
- Assessment of p53-deficient neurons for comparison.
Main Results:
- Quantification of 150 protein expression changes in wild-type neurons post-DNA damage.
- Identified perturbations in mitochondrial function, free radical production, and neuritogenesis, which were p53-dependent.
- Observed protein changes in Tau and cofilin, mirroring neurodegenerative abnormalities.
- Demonstrated a p53-dependent decrease in protein kinase A (PKA) signaling pathway members.
- Showed that PKA inhibition promotes neuronal death independently of DNA damage.
Conclusions:
- High-throughput mass spectrometry is a powerful tool for quantitative neuronal proteome analysis.
- DNA damage induces p53-dependent proteomic alterations that contribute to neuronal death and resemble neurodegenerative conditions.
- Down-regulation of PKA signaling represents a novel mechanism in p53-dependent neuronal death.
