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. 2015 Feb;93(2):199-214.
doi: 10.1002/jnr.23503. Epub 2014 Nov 14.

A Systems Biology Strategy to Identify Molecular Mechanisms of Action and Protein Indicators of Traumatic Brain Injury

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Free PMC article

A Systems Biology Strategy to Identify Molecular Mechanisms of Action and Protein Indicators of Traumatic Brain Injury

Chenggang Yu et al. J Neurosci Res. .
Free PMC article

Abstract

The multifactorial nature of traumatic brain injury (TBI), especially the complex secondary tissue injury involving intertwined networks of molecular pathways that mediate cellular behavior, has confounded attempts to elucidate the pathology underlying the progression of TBI. Here, systems biology strategies are exploited to identify novel molecular mechanisms and protein indicators of brain injury. To this end, we performed a meta-analysis of four distinct high-throughput gene expression studies involving different animal models of TBI. By using canonical pathways and a large human protein-interaction network as a scaffold, we separately overlaid the gene expression data from each study to identify molecular signatures that were conserved across the different studies. At 24 hr after injury, the significantly activated molecular signatures were nonspecific to TBI, whereas the significantly suppressed molecular signatures were specific to the nervous system. In particular, we identified a suppressed subnetwork consisting of 58 highly interacting, coregulated proteins associated with synaptic function. We selected three proteins from this subnetwork, postsynaptic density protein 95, nitric oxide synthase 1, and disrupted in schizophrenia 1, and hypothesized that their abundance would be significantly reduced after TBI. In a penetrating ballistic-like brain injury rat model of severe TBI, Western blot analysis confirmed our hypothesis. In addition, our analysis recovered 12 previously identified protein biomarkers of TBI. The results suggest that systems biology may provide an efficient, high-yield approach to generate testable hypotheses that can be experimentally validated to identify novel mechanisms of action and molecular indicators of TBI.

Keywords: biomarkers; pathway analysis; protein-protein interaction networks; systems biology; traumatic brain injury.

Figures

Fig 1
Fig 1
Illustration of the systems biology strategy, in which we used computational analysis to generate testable hypotheses that can be experimentally validated in the laboratory. We started by performing a meta-analysis of four gene expression data sets and separately overlaying each data set onto two types of biological networks, canonical molecular pathways and PPI networks. In these analyses, we considered up- and downregulated genes separately because previous findings support the hypothesis that biological processes are characterized by interacting, coregulated proteins. Next, we identified statistically significant molecular mechanisms of action and protein indicators of TBI that were conserved across the studies and analyses. Finally, we experimentally tested protein indicators of TBI with an in vivo animal model. The right side of the figure illustrates the construction of a PPI subnetwork (shaded area) formed by three proteins (red circles, denoting module centers C1, C2, and C3 from three protein modules) and three other proteins (green circles) from these modules. KEGG, Kyoto Encyclopedia of Genes and Genomes.
Fig 2
Fig 2
Significantly regulated pathways conserved in at least three of the four data sets.
Fig 3
Fig 3
Significantly regulated protein–protein interaction (PPI) modules conserved in at least two of the four data sets.
Fig 4
Fig 4
A: The six suppressed PPI modules, each composed of a module center protein (red hexagon) and other proteins directly connected to the module center, for a total of 296 interacting proteins. B: Synaptic subnetwork extracted from the six modules in A consists of 58 proteins, including the six module center proteins and proteins that interacted with two or more module centers (green circles in A). Forty-five percent (26 of 58, corresponding to P < 2.0 × 10−20) of these proteins were associated with synapses and 28% (16 of 58, corresponding to P < 8.0 × 10−3) with schizophrenia. *Proteins selected for experimental testing.
Fig 5
Fig 5
Temporal profile of the aggregate expression scores of the genes in the synaptic subnetwork (Fig. 4B) for data set R-FPIm in Table1. The gene expression was suppressed during the 21-day period, reaching a nadir between 24 hr and 72 hr after injury and eventually returning to baseline at 21 days.
Fig 6
Fig 6
Western blot analyses of the three proteins hypothesized to have a reduced abundance at 24 hr after PBBI compared with sham (craniotomy surgery without PBBI); n = 3 animals for PBBI and sham unless otherwise noted. A: DISC1 in ipsilateral tissues shows reduced abundances (**P < 0.001). 1For ipsilateral and contralateral tissues, each bar in the graphs represents the sum of all bands on one gel lane for one animal. B: PSD95 in ipsilateral tissues also shows reduced abundances compared with sham (*P < 0.05). Unexplained increased abundances in contralateral tissues were not significant (P > 0.05). C: NOS1 shows reduced abundances in both ipsilateral tissues (*P < 0.02) and contralateral tissues (*P < 0.005). 2One PBBI sample and one sham sample yielded no signal.
Fig 7
Fig 7
Genes in the significant pathways and in the significant subnetworks had greater overlap across the four data sets than equal-sized sets of top-ranked, differentially expressed individual genes in the data sets. We also computed the overlap of equal-sized randomly selected individual genes from each data set (1,000 realizations; error bar represents one standard deviation). A,B: Analysis of activated and suppressed pathways, respectively. C,D: Analysis of activated and suppressed subnetworks, respectively.

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