Intranasal delivery of HMGB1-binding heptamer peptide confers a robust neuroprotection in the postischemic brain

doi:10.1016/j.neulet.2012.07.040

Neuroscience Letters

Volume 525, Issue 2, 13 September 2012, Pages 179-183

https://doi.org/10.1016/j.neulet.2012.07.040 Get rights and content

Abstract

High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In a previous report, we showed that HMGB1 is massively released during NMDA-induced acute damaging process in the postischemic brain and triggers inflammatory processes and induces neuronal apoptosis. We have also reported a robust neuroprotection of intranasally delivered HMGB1 siRNA in the postischemic rat brain (middle cerebral artery occlusion (MCAO), 60 min). In the present study, we investigated the therapeutic efficacy of intranasally delivered HMGB1 binding heptamer peptide (HBHP; HMSKPVQ), which was selected using a phage display approach, in the same stroke animal model. A pull-down assay using biotin-labeled HBHP showed that HBHP binds directly to HMGB1, specifically to HMGB1 A box, confirming HMGB1/HBHP interaction. HBHP significantly suppressed HMGB1-mediated neuronal cell death in primary cortical cultures and HMGB1/HBHP binding was detected in NMDA-conditioned culture media. However, a heptamer peptide composed of a scrambled sequence of the seven amino acids in HBHP failed to bind HMGB1 and had no protective effect. Furthermore, HBHP (300 ng) delivered intranasally at 30 min before MCAO significantly suppressed infarct volume in the postischemic rat brain (maximal reduction by 41.8 ± 5.4%) and ameliorated neurological and behavioral deficits. In contrast, scrambled heptamer peptide had no protective effect at the same dose. Together these results suggest that intranasal HBHP ameliorates neuronal damage in the ischemic brain by binding HMGB1, which might inhibit the function of HMGB1 as an endogenous danger signal molecule.

Highlights

► HBHP is a heptamer peptide selected as a HMGB1 binding peptide using a phage display approach. ► HBHP was confirmed to bind A box region of HMGB1 in a pull-down assay. ► HBHP suppressed HMGB1-mediated neuronal cell death in primary cortical cultures. ► Intranasally delivered HBHP has a robust neuroprotective effect in the ischemic brain.

Introduction

High mobility group box 1 (HMGB1), an endogenous danger signal molecule, is released by necrotic cells into the extracellular milieu or is actively secreted by macrophages and monocytes and induces inflammation [2], [16]. HMGB1 secretion does not proceed via the classical endoplasmic reticulum–Golgi pathway, but the phosphorylation and acetylation of HMGB1 are known to be involved in its translocation from the nucleus to cytoplasm. Accumulating evidences indicate that HMGB1 forms complexes with various molecules, including LPS, IL-1β, and ssDNA, and it acts as an inflammatory inducer or booster by binding with several receptors, including Toll-like receptors (TLRs) [7], [17], [19].

In the brain, HMGB1 is released under various pathological conditions and then participates in inflammatory processes [1]. In a previous report, we found that HMGB1 is massively released by NMDA-induced acute damage in the postischemic brain, and that it then exacerbates neuronal damage and triggers inflammatory processes [11], [12]. We also reported that siRNA-mediated HMGB1 knockdown and HMGB1 A box-mediated inhibition of HMGB1 markedly reduced infarct volume in middle cerebral artery occlusion (MCAO) animal model [8], [10], [12]. In addition, the administration of HMGB1 monoclonal antibody has been reported to ameliorate brain infarctions [14] and to protect blood–brain barrier [20] after transient ischemia.

In the present study, we investigated the neuroprotective effects of HMGB1 binding heptamer peptide (HBHP; HMSKPVQ) in the postischemic rat brain. HBHP is one of many peptides found to bind HMGB1 A box by ligand selection screening using a large library of heptapeptides displayed on phages [4]. HMGB1/HBHP binding was confirmed by a pull-down assay and neuroprotective potency of HBHP was determined in the ischemic brain after intranasal administration before or after MCAO. Furthermore, we sought to elucidate the molecular mechanism responsible for its neuroprotective effects.

Section snippets

Materials and methods

Middle cerebral artery occlusion (MCAO) was carried out as previously described [12]. In brief, male Sprague-Dawley rats (250–300 g) were anesthetized with 5% isoflurane in a 30% oxygen/70% nitrous oxide gas mixture and maintained using 0.5% isoflurane in the same gas mixture throughout the procedure. MCA occlusion was maintained for 1 h using a nylon suture, and this was followed by reperfusion for 2 days. During the procedure, the left femoral artery was cannulated to obtain blood samples and

Results

Biotin pull-down assays were used to examine the interaction between HBHP (HMSKPVQ) and HMGB1. Recombinant HMGB1 was incubated with biotin-tagged HBHP and precipitated with streptavidin beads, and HBHP/HMGB1 binding was examined by immunoblotting using anti-HMGB1 antibody. Recombinant HMGB1 bound strongly with HBHP in a dose-dependent manner (Fig. 1A) but not with HBHP-sc (PMQSKHV), containing a scrambled sequence of the same amino acids in HBHP (Fig. 1B). When 5 μg/ml of recombinant HMGB1 was

Discussion

When released extracellularly, HMGB1 serves as a danger signal that evokes inflammatory reactions by activating various immune-related cells, which includes microglia in the brain [12]. In our previous studies, we found HMGB1 levels in serum [12] and CSF [10] were rapidly increased from 3 h post-MCAO until 7 days post-MCAO. In addition, we showed that extracellular HMGB1 released from damaged neurons in brain activates microglia [12] and induces the apoptosis of neighboring neurons [11].

Acknowledgments

This work was financially supported by Translational Research Grant (A100793) funded by Korea Health Industry Development Institute (KHIDI) for J-K Lee.

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Intranasal delivery of HMGB1-binding heptamer peptide confers a robust neuroprotection in the postischemic brain

Abstract

Highlights

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

Cytokine

Journal of Biological Chemistry

Blood

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Neurobiology of Disease

Molecular Therapy

Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion

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Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats

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HMGB1 inhibits macrophage activity in efferocytosis through binding to the alphavbeta3-integrin

American Journal of Physiology—Cell Physiology

Receptor for advanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis

Cancer Research