Pathway analysis reveals common pro-survival mechanisms of metyrapone and carbenoxolone after traumatic brain injury

Abstract

Developing new pharmacotherapies for traumatic brain injury (TBI) requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT) and carbenoxolone (CB), which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11β hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks). We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that suppressing expression of potentially harmful genes, and also, surprisingly, reduced expression of pro-survival genes may be a hallmark of neuroprotective therapeutic effects.

Figure 1. Neuroprotective effects of metyrapone (MT) or carbenoxolone (CB) treatment after traumatic brain injury (TBI).

(A,B) Treatment of TBI rats with MT or CB significantly reduced numbers of degenerating, FJ-positive neurons in the CA3 subfield of the rat hippocampus. n = 6 per group, *p<0.05 (Bonferroni-Dunn test), CA (Cornu Ammonis). (C) Heat map of 870 genes >2-fold showing that both MT and CB treatment attenuated TBI-induced gene expression in hippocampal CA3 pyramidal neurons, in many cases returning gene expression levels to that of sham-operated control animals. (D) Both MT and CB prevented the TBI-induced increase in expression of 11βHSD1 (11β hydroxysteroid dehydrogenase type 1) in surviving, Fluoro-Jade-negative hippocampal neurons. (E) Although very different compounds, the transcriptional profiles induced by MT or CB are remarkably similar. Both drugs attenuate expression of several common genes in a custom pathway of TBI-induced genes. (See Fig. S15 for symbol key).

Figure 2. Common genes in cell death and stress response networks are affected by metyrapone or carbenoxolone.

(A) Apoptosis signaling at 24 h post-TBI. (B) Death receptor signaling at 4 h post-TBI. (C) Corticotropin-releasing hormone (CRH) signaling at 4 h post-TBI. (D) Amyloid processing pathway at 4 h post-TBI. Gene expression in all groups for pathways A–C is shown up or down-regulated relative to sham control levels with no fold cut-offs. Metyrapone and carbenoxolone attenuate expression of both pro-death and pro-survival genes in these pathways such as FLIP, which is known to protect against ischemic cell death. (See Fig. S15 for symbol key).

Figure 3. Metyrapone and carbenoxolone exert similar effects on several common gene targets in prosurvival pathways.

(A) Nuclear factor of activated T cells (NFAT) signaling at 24 h post-TBI. (B) Cyclic AMP-response element binding (CREB) protein signaling at 4 h post-TBI. (C) Protein kinase A (PKA) signaling at 4 h post-TBI. These signaling pathways are associated with cell survival, synaptic plasticity, development and regeneration. (See Fig. S15 for symbol key).

Figure 4. Metyrapone and carbenoxolone have similar effects on the pro-survival Phosphatidylinositol 3 kinase/AKT (PI3K/AKT) pathway.

Gene expression is shown at 4 h (2 fold cut-off) and 24 h post-TBI (no cut-off). A key cell survival associated gene, NFκB1A, is upregulated by both drugs at 24 h compared with TBI alone. (See Fig. S15 for symbol key).

Figure 5. Metyrapone and carbenoxolone attenuate gene expression in both pro-survival and pro-death pathways.

(A) Axon guidance signaling pathway is associated with regeneration and growth. (B) TNFR1 signaling pathway is associated with inflammation and cell death. (See Fig. S15 for symbol key).

Figure 6. Metyrapone and carbenoxolone attenuate gene expression in pathways associated with production of reactive oxygen species (ROS).

(A) Both drugs downregulate expression of subunits of NADH dehydrogenase in Complex 1 (primary source of reactive oxygen species produced in mitochondria), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V) in the mitochondrial oxidative phosphorylation pathway at 4 h post-TBI. (B) Expression of multiple genes in the canonical Nitric oxide and Reactive Oxygen Species (NO & ROS) in macrophages pathway at 4 h post-TBI (2 fold cut-off) are also downregulated after metyrapone or carbenoxolone treatment suggesting a drug-induced reduction in oxidative stress. (See Fig. S15 for symbol key).