A novel function of neuroglobin for neuroregeneration in mice after optic nerve injury

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Highlights

  • A novel function of Ngb for neuroregeneration.

  • Mammalian Ngb has neuroprotective and neuroregenerative properties.

  • Ngb injection into mouse eye promotes cell survival and axonal regeneration in vivo.

Abstract

Neuroglobin (Ngb) is a recently discovered heme protein in the vertebrate brain that can bind to oxygen molecules. Mammalian Ngb plays a crucial role in neuroprotection under conditions of oxidative stress. To investigate other potential functions of Ngb, we investigated the mouse retinal Ngb system following optic nerve injury. In the retina of control mice, Ngb immunoreactivity was limited to the retinal ganglion cell (RGC) layer, and this immunoreactivity rapidly decreased to less than 50% of the control level 5 days after optic nerve injury. On the basis of this decrease, we designed in vivo experiments with enhanced expression of Ngb using adult mouse retina. The enhanced expression of Ngb was achieved by injecting chimeric human Ngb protein, which included the cell membrane-penetrating module of fish Ngb. One-day pretreatment with chimeric Ngb increased immunoreactivity levels of Ngb two-fold in mouse RGCs and increased the number of surviving RGCs three-fold by 14 days after optic nerve injury compared with vehicle controls. Furthermore, in the mouse retinas showing enhanced Ngb expression, several regenerating central optic axons exhibited outgrowth and were found to pass through the nerve crush site 14 days after nerve injury. No such regenerating optic axons were observed in the control mouse optic nerve during the same time frame. The data obtained from in vivo experiments strongly indicate that mammalian Ngb has neuroprotective and neuroregenerative properties.

Introduction

Neuroglobin (Ngb) is the third type of heme protein to be discovered after hemoglobin and myoglobin. It was originally identified in the mammalian brain and has the ability to bind to oxygen (O2) molecules [1]. Because of the low concentration of Ngb in brain tissue, Ngb likely does not serve as an O2 carrier; rather, it has neuroprotective properties under conditions of oxidative stress, such as those caused by ischemia, reperfusion in vitro [2], [3], and reperfusion in vivo [4]. Although Ngb was initially identified in mammalian species, it is also present in non-mammalian vertebrate species such as the zebrafish [5], [6]. Human and zebrafish Ngb proteins share about 50% of their amino acid sequence identity, and their associated genes comprise four exons interrupted by three introns [5], [6], [7].

Mammalian Ngb displays guanine nucleotide dissociation inhibitor (GDI) activity that is tightly correlated with its neuroprotective properties [8], [9], [10], [11], [12]. In contrast, zebrafish Ngb does not show similar GDI activity but instead presents a cell membrane-penetrating module [13], [14], [15], [16]. Given these distinct properties, we constructed chimeric human Ngb by replacing a human module with a zebrafish cell membrane-penetrating module to produce a recombinant cell membrane-penetrating protein of human Ngb [14], [16].

Over the past two decades, our laboratory has identified many nerve regeneration-associated genes (RAGs) in the fish visual system. Generally, fish central nervous system (CNS) neurons can regenerate even after nerve transection, whereas mammalian CNS neurons cannot [17], [18], [19]. We have previously cloned RAGs from the axotomized zebrafish visual system, including the retina, and found that they are largely classified into two groups: one that displays anti-oxidant or anti-apoptotic activity and another that displays neurite outgrowth activity [19], [20], [21], [22], [23], [24], [25], [26]. The former type can be induced at an early stage of nerve regeneration immediately after optic nerve injury, whereas the latter can typically be induced at a later stage of nerve regeneration [19]. Moreover, we have compared the behavior of zebrafish and mammalian homolog RAGs after optic nerve injury. Interestingly, although RAG genes in mice are maximally expressed in the normal retina, expression rapidly decreases immediately after optic nerve injury, occasionally completely disappearing. In a previous study, we found that Ngb mRNA and Ngb proteins were transiently upregulated in the zebrafish retina 3 days after optic nerve injury, only to return to control levels 7 days later [16]. In vitro studies have shown that overexpression of mouse Ngb protein enhances cell viability under hypoxia/reoxygenation conditions [27]. Additionally, we have confirmed that chimeric human Ngb promotes neurite outgrowth activity in rat pheochromocytoma PC12 cells [28].

To investigate the neuroprotective and neuroregenerative properties of mammalian Ngb, we investigated the mouse retinal Ngb system both with and without chimeric cell membrane-penetrating human Ngb in vivo.

Section snippets

Animals

All animals were maintained and handled in accordance with the Guiding Principles in the Care and Use of Animals and the Kanazawa University's guidelines for animal experiments. Male C57BL/6 mice (8–9 weeks old; Japan SLC, Inc., Shizuoka, Japan) were reared in clear plastic cages and kept under a 12 h light–dark cycle at 23 °C. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30–40 mg/kg body weight). The optic nerve was crushed using forceps 1 mm posterior to the

Reduction of Ngb in mouse retina after optic nerve injury

Ngb is specifically expressed in the vertebrate CNS and peripheral nervous systems, with the retina having a 100-fold higher concentration than other CNS tissues [34].

To characterize how Ngb concentration is altered in mouse retina following optic nerve injury, we performed Ngb immunohistochemical staining at various times after nerve injury (Fig. 1). Ngb was localized in all nuclear layers, especially in the ganglion cell layer (GCL) in control mice retina (Fig. 1A, 0 d). These Ngb signals in

Comparative studies of the Ngb system in the mouse and zebrafish retina

Previously, we found that in the zebrafish retina, Ngb mRNA is expressed in amacrine cells, whereas Ngb proteins are expressed in both amacrine cells and the inner plexiform layer [16]. In the present study, we confirmed that Ngb proteins are only expressed in the RGCs of the mouse retina [27], [38]. The localization of Ngb proteins in the inner plexiform layer of the zebrafish is easily imaged due to the secretion of Ngb from the amacrine cells and the cell membrane-penetrating module of

Acknowledgement

This work was supported by Grants-in-Aid for Scientific Research to K.S. (No. 26350958 and No. 17K01945), Y.K. (No. 22791651 and No. 16K11335) and K.W. (No. 17K07329) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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    These authors contributed equally to this work.

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