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New biomarkers that distinguish acute from chronic phases of traumatic brain injury (TBI) have been identified by scientists at Arizona State University (ASU). Their work could lead to new therapeutics and diagnostics and also help explain why people who have had TBI are more susceptible to developing neurodegenerative diseases such as Parkinson’s and Alzheimer’s later in life.

The team found that in the acute phase markers were associated with mainly metabolic and mitochondrial dysfunction, whereas, the chronic TBI motif was largely associated with neurodegenerative processes.

For this work, the team used a unique discovery pipeline that combines, “in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis.”  The work was led by Sarah Stabenfeldt, PhD, an ASU professor and corresponding author of the study. It appears in Science Advances.

“Unfortunately, the molecular and cellular mechanisms of TBI injury progression are multifaceted and have yet to be fully elucidated,” said Stabenfeldt. “Consequently, this complexity affects the development of diagnostic and treatment options for TBI; the goal of our research was to address these current limitations.”

TBI is a growing public health concern, affecting more than 1.7 million Americans at an estimated annual cost of $76.5 billion dollars. It is a leading cause of death and disability for children and young adults in industrialized countries, and people who experience TBI are more likely to develop severe, long-term cognitive and behavioral deficits. It is a particular issue among athletes who participate in sports that put them at high risk for concussion, which has led to growing interest in biomarkers for this condition.

Novel biomarker discovery pipeline

Stabenfeldt’s team used what they call a “bottom-up” approach to biomarker discovery.

“‘Top-down’ discovery methods are focused on assessing candidate biomarkers based on their known involvement in the condition of interest,” said study first author Briana Martinez, a recent PhD graduate in Stabenfeldt’s lab. “In contrast, a ‘bottom-up’ method analyzes changes in tissue composition and finds a way to connect those changes to the condition. It’s a more unbiased approach but can be risky because you can possibly identify markers that are not specific to the condition or pathology of interest.”

Starting with a mouse model, they used several “biopanning” tools and techniques to identify and capture molecules, including a “bait” technique for fishing out potential target molecules using a phage-display system, high-speed DNA sequencing to identify protein targets within the genome, and mass-spectrometry to sequence the peptide fragments from the phase display experiments.

When a TBI occurs, the initial injury can disrupt the blood-brain barrier (BBB), which triggers a cascade of cell death, torn, disrupted tissues, and debris. The long-term injury causes inflammation and swelling, and results in the immune response to spring into action, but also can lead to an impairment of the brain’s energy sources, or can choke off the brain’s blood supply, leading to more neuronal cell death and permanent disability.

A key advantage of ASU’s suite of experimental tools and techniques of the phage display system is that the molecules and potential biomarkers identified are small enough to slip through the tiny holes within the meshwork of the BBB—thus, opening the way to therapeutics based on these molecules.

“Our study leverages the sensitivity and specificity of phage to discover novel targeting motifs,” said Stabenfeldt. “The combination of phage and NGS [next-generation sequencing] has been used previously, thereby leveraging bioinformatic analysis. The unique contribution of our study is putting all of these tools together specifically for an in vivo model of TBI.”

 

 

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