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. 2015 Aug;134(4):677-92.
doi: 10.1111/jnc.13176. Epub 2015 Jun 19.

The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming and CREB pathway up-regulation

Dong Liu  1 , Yongqing Zhang  2 , Robert Gharavi  1 , Hee Ra Park  3 , Jaewon Lee  3 , Sana Siddiqui  1 , Richard Telljohann  1 , Matthew R Nassar  1 , Roy G Cutler  1 , Kevin G Becker  2 , Mark P Mattson  1
Affiliations

The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming and CREB pathway up-regulation

Dong Liu et al. J Neurochem. 2015 Aug.

Abstract

Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4-dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca(2+) levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP-treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin--PI3K - MAPK pathways, and up-regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up-regulated in response to DNP. CREB (cAMP-response element-binding protein) signaling, Arc and brain-derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up-regulated in response to DNP, and DNP-treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP-induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up-regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity. Physiological bioenergetic challenges such as exercise and fasting can enhance neuroplasticity and protect neurons against injury and neurodegeneration. Here, we show that the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) elicits adaptive signaling responses in the cerebral cortex involving activation of Ca(2+) -CREB and autophagy pathways, and inhibition of mTOR and insulin signaling pathways. The molecular reprogramming induced by DNP, which is similar to that of exercise and fasting, is associated with improved learning and memory, suggesting potential therapeutic applications for DNP.

Keywords: 2,4-dinitrophenol; BDNF; CREB; autophagy; insulin signaling; mTOR.

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Conflict of interest statement

Conflicts of interests

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Evidence that mild mitochondrial uncoupling modulates mTOR signaling in the cerebral cortex. (a and b) Schematic diagrams depicting the effects of DNP on the expression of genes in the mTOR signaling pathway showing genes upregulated (red) and downregulated (green) in the cerebral cortex at 24 hours (a) or 72 hours (b) following DNP treatment compared to vehicle-treated control mice. Grey denotes no significant change and white denotes data not available. Genes with a z-score above 1.5 compared to vehicle-treated control were considered significantly changed. (c) Immunoblot analysis of the indicated proteins in cerebral cortex samples from DNP-treated and control mice (n= 7–10 mice/group; samples from 3 animals/group were presented). (d and e). Results of densitometric analysis of the indicated proteins (normalized to β-actin level). Values are mean ± SD. *p<0.05 two tailed compared to the values of DNP to vehicle-treated control mice.
Figure 2
Figure 2
DNP treatment triggers mTORC1 and mTORC2 signaling network re-programming in the cerebral cortex. (a) Gene expression network profile showing genes in the mTORC1 signaling network that were upregulated (red) or downregulated (green) in the cerebral cortex of mice in response to DNP treatment (72 hours). (b) Gene expression profile showing upregulated (red) and downregulated (green) genes in the mTORC2 signaling network from cerebral cortex of mice in response to DNP treatment (72 hours). The attributes that appear both red and green are genes that encode proteins that form complexes; some genes that encode proteins in the complex are up-regulated (red) and some are down-regulated (green). (c) List of genes with z-ratio change in response to DNP treatment in mTORC1 signaling network at 72 hours. (d) List of genes with z-ratio change in response to DNP treatment in mTORC2 signaling network at 72 hours. (e) Immunoblot showing levels of FoxO1, FoxO3a, SGK1 and β-actin in cerebral cortex tissue samples from vehicle-treated control mice and mice that had been treated with DNP for the indicated time periods. (f and g) Densitometric quantification of protein band intensities normalized to β-actin. Values are mean ± SD.
Figure 3
Figure 3
DNP treatment reduces expression of genes in the insulin signaling pathway in the cerebral cortex. (a) Schematic diagram on gene expression in the insulin signaling pathway showing genes upregulated (red) and downregulated (green) in the cerebral cortex within 6 hours of DNP treatment. (b) Genes changed in insulin signaling pathways in the cerebral cortex at 6, 24 and 72 hours following DNP treatment. (c) Immunoblot showing levels of p-Akt, total Akt, p-ERK, total ERK and actin in cerebral cortex tissue samples from vehicle-treated control mice, and mice that had been treated with DNP for the indicated time periods. (d and e) Densitometric quantification of Akt and p-Akt (D), and Erk1/2 and p-Erk1/2 (E) protein band intensities normalized to β-actin. Values are mean ± SD *p<0.05 compared to the control value.
Figure 4
Figure 4
DNP treatment engages Ca2+ and CREB signaling, up-regulates synaptic plasticity-related genes and enhances memory consolidation. (a) Schematic diagram of Ca2+ and CREB signaling pathways showing genes upregulated (red) and downregulated (green) in the cerebral cortex of mice treated with DNP (72 hours). (b) Immunoblot showing levels of p-CREB, total CREB and Arc/Arg3.1 in cerebral cortex tissue samples from vehicle-treated control mice, and mice that had been treated with DNP for the indicated time periods. (c and d) Densitometric quantification of CREB, p-CREB and Arc/Arg3.1 protein band intensities normalized to β-actin. Values are mean ± SD (n = 7 mice per group). *p<0.05; **p<0.01. (e and f) Parametric analysis of gene set enrichment (PAGE) revealed genes in the CREB signaling pathway, Bdnf and Arc/Arg3.1 upregulated in the cerebral cortex in response to DNP treatment (6 hour time point). Data represent comparisons of 3 control and 3 DNP-treated mice in (c). (g and h) Real-time PCR results show levels of Arc/Arg3.1 and Bdnf mRNAs in cerebral cortex samples from control and DNP-treated mice. (i) Passive avoidance testing was performed to assess memory retention. There was no difference in latency time between vehicle and DNP-treated mice during training. DNP-treated mice exhibited significantly greater retention of the memory of the shock when tested 24 hours after training. Values are mean ± SD (4 mice in the vehicle group and 5 mice in the DNP group). *p<0.05 vs. vehicle (ANOVA with Fisher’s PLSD procedure).
Figure 5
Figure 5
Evidence that mitochondrial uncoupling upregulates autophagy in the cerebral cortex. (a) Relative changes in the expression of genes involved in autophagy, mitophagy and mitochondrial dynamics from the cerebral cortex of mice in response to DNP treatment for 6 hours. (b) Representative immunoblots showing relative levels of the indicated proteins in mouse cerebral cortex samples from the indicated groups. (c) Densitometric analysis of LC3-I, LC3-II and beclin proteins normalized to β-actin. (d) LC3-II/LC3-I ratio in cerebral cortex of mice treated with DNP for the indicated time periods. (e) Densitometric analysis of Drp1 and Hsp70 protein levels in and the cerebral cortex of mice in the indicated treatment groups (n = 7 mice/group). *p<0.05.
Figure 6
Figure 6
Model for brain signaling network reprogramming in response to mild mitochondrial uncoupling. Mild mitochondrial uncoupling induces a proton leak and enhances energy expenditure. Three major interactive signaling pathways affected by mitochondrial uncoupling are the mTOR and insulin signaling pathways, which are downregulated, and the Ca2+ – CamKII – CREB and autophagy pathways which is upregulated. These changes bolster cellular adaptive stress resistance, and enhance synaptic plasticity by upregulation of Arc/Arg3.1 and BDNF gene transcription. Abbreviations: TSC1/TSC2, tuberous sclerosis complexes 1 and 2; mTOR, mammalian target of rapamycin; CREB, cAMP-response element binding protein; Rheb, Ras homolog enriched in brain; BDNF, brain-derived neurotrophic factor; S6K, ribosomal S6 kinase.
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