Elsevier

Clinical Biochemistry

Volume 48, Issues 13–14, September 2015, Pages 843-848
Clinical Biochemistry

Serum neurogranin measurement as a biomarker of acute traumatic brain injury

https://doi.org/10.1016/j.clinbiochem.2015.05.015Get rights and content

Highlights

  • Developed a highly sensitive and reproducible ELISA for neurogranin.

  • Serum neurogranin levels in TBI patients are significantly higher than those in controls.

  • Neurogranin may be a biomarker for acute TBI diagnosis and recovery.

Abstract

Objectives

Neurogranin (NRGN) is a small neuronal protein that plays an important role in synaptic signaling by regulating calmodulin (CaM) availability. In this study, we developed an ELISA to measure NRGN quantitatively in serum samples from a cohort of acute traumatic brain injury (TBI) patients and a non-TBI control cohort, and explored the potential value of NRGN as a circulating biomarker for TBI.

Design and methods

Recombinant His-NRGN protein was used to develop mouse monoclonal capture and rabbit polyclonal detection antibodies, and they were used to develop a sandwich ELISA. After validation, we used this ELISA to measure serum samples from a cohort of typical adult acute TBI patients (N = 76 TBI cases) and non-TBI control patients (N = 150 controls).

Results

The NRGN ELISA lower limit of detection was 0.055 ng/mL, lower limit of quantification was 0.2 ng/mL, and interassay CVs were ≤ 10.7%. The average recovery was 99.9% (range from 97.2–102%). Serum NRGN concentrations in TBI cases were significantly higher than in controls (median values were 0.18 ng/mL vs. 0.02 ng/mL, p < 0.0001), but did not discriminate TBI cases with intracranial hemorrhage (p = 0.09).

Conclusions

We have developed a highly sensitive and reproducible ELISA for measuring circulating NRGN in blood samples. Serum NRGN concentrations in acute TBI patients were significantly higher than in controls, indicating that NRGN could have utility as a circulating biomarker for acute TBI. This report provides evidence to support larger and controlled TBI clinical studies for NRGN validation and prediction of outcomes.

Introduction

Neurogranin (NRGN or Ng) is a highly conserved (+ 96% protein sequence homology among mammals) small neuronal protein (78 AAs, ~ 7.6 kDa) originally identified in rat brain [1], [2], [3]. It binds to calmodulin (CaM) and serves as a substrate for protein kinase C (PKC) [2], [4], [5], [6], [7], from where its another name BICKS (B-50 immunoreactive C kinase substrate) originated [8]. NRGN protein is found in the cell bodies of cerebral cortex neurons in layers II–VI, and in apical and basal dendrites of pyramidal neurons [9]. It is concentrated in dendritic spines where it participates in the synaptic signaling events by regulating the availability of calmodulin (CaM) [2], [10]. NRGN plays an important role in synaptic plasticity and cognitive function where it serves as a key second messenger in mediating the effects of thyroid hormone on the brain [11]. Although NRGN knockout mice display a structurally normal phenotype, they have a severe functional impairment of spatial learning and a decrease in long term potentiation (LTP) induction, most likely due to the defective activation of calcium/CaM kinase II (CaMKII) autophosphorylation [12]. Cognitive, learning and memory deficits have been reported after traumatic brain injury (TBI), however, the mechanisms underlining these deficits are not well understood [13]. As NRGN is also a small protein, it may be able to cross an intact and or damaged blood brain barrier with relative ease, therefore, we hypothesize that NRGN may serve as a circulating biomarker of acute TBI. In this report we describe the development of a NRGN ELISA and the diagnostic accuracy of circulating NRGN in distinguishing between acute TBI cases and non-TBI controls in the Emergency Department.

Section snippets

Recombinant NRGN protein production

The construction of the expression vector for His tagged NRGN and the recombinant NRGN protein production has been previously described [14]. Briefly, we purified cDNA fragment of full-length human NRGN gene by restriction enzyme digestion (Sgf I and Mlu I) and agarose gel purification from human cDNA ORF clone (RC201209, Origene, Rockville, MD), and then ligated into bacterial expression vector pEX-N-His (PS100030, Origene, Rockville, MD) with the same restriction enzymes sites. The DNA

Expression construct and recombinant protein production

To develop antibodies for NRGN, we cloned the entire human NRGN coding region into pEX-N-His expression vector using Sgf I and Mlu I restriction enzyme sites, giving an N-terminal 6XHis tagged NRGN protein. His-NRGN recombinant protein was extracted from E. coli lysate in native condition and affinity purified on a Ni-NTA beads and dialyzed against 1 × PBS. The purified His-NRGN recombinant protein migrated in SDS-PAGE at approximately 13 kDa (Fig. 1) as a result of the addition of His tag. The

Discussion

In this study, we developed a sensitive NRGN sandwich ELISA with initial evidence that NRGN deserves further investigation as a candidate biomarker of acute TBI. The human NRGN gene is mapped to chromosome 11q24, spans ~ 12.5 kb and contains 4 exons and 3 introns, which encodes a 78-amino acid protein product [16]. NRGN gene expression in brain starts at embryonic day 18 (E18) in the rat, following another 2 peaks of protein expression at postnatal day 14 and 20 respectively. These developmental

Conclusion

We have developed a new and sensitive ELISA for the neuronal protein NRGN, that demonstrates significant TBI diagnostic ability. Considering NRGN's pivotal role in learning, NRGN may play an important role as a biomarker of acute TBI and recovery.

    Abbreviations

    Neurogranin

    (NRGN)

    Calmodulin

    (CaM)

    CaM kinase II

    (CaMKII)

    Traumatic brain injury

    (TBI)

    American College of Emergency Physicians

    (ACEP)

    Emergency Department

    (ED)

    Area under the receiver operator curve

    (AUC)

Acknowledgments

We thank the Monoclonal Antibody Core Facility (MACF) at Johns Hopkins University for generating the monoclonal anti-neurogranin antibody. Under a licensing agreement between ImmunArray and the Johns Hopkins University, Drs. Everett, Yang and Korley are entitled to royalties on an invention described in this article.

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