Expression and localization of the iron–siderophore binding protein lipocalin 2 in the normal rat brain and after kainate-induced excitotoxicity

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Abstract

Lipocalin 2 (LCN2) is produced by mammalian hosts to bind bacterial siderophore and sequester free iron as part of an innate immune response, and could also play a role in tissue iron homeostasis, but thus far, little is known about its expression in the CNS. The present study was carried out to study the expression of the lipocalin in the normal rat brain and after neuronal injury induced by kainate (KA). Low levels of LCN2 mRNA and protein expression were detected in most regions of the normal brain except the olfactory bulb, brainstem and cerebellum. KA lesions resulted in damage to the hippocampus, leading to an early increase at three days and a sustained elevation in LCN2 mRNA level of 16-fold, and protein expression at 80-fold in the lesioned tissue compared to controls at 2 weeks post-KA injection. The sustained elevation in mRNA expression was not detected among other lipocalins surveyed using real-time RT-PCR – apoD, PGDS, Rbp4 and LCN5. Single and double immunostaining confirmed that LCN2 is present in astrocytes in the olfactory bulb, brainstem and cerebellum of the normal brain, and reactive astrocytes in the KA-lesioned hippocampus. In conclusion, the present study showed LCN2 to be present in select brain regions, and is upregulated in astrocytes after neuronal injury induced by kainate. We postulate that, as in the periphery, LCN2 may have a role in iron transport or trafficking in the CNS.

Highlights

Lipocalin 2 (LCN2) is expressed in astrocytes and the choroid plexus of normal brains. ► Increased LCN2 expression is observed in astrocytes after kainate-induced excitotoxicity. ► LCN2 may affect brain iron metabolism through binding to iron–siderophore complexes.

Introduction

Lipocalins are a diverse family of secreted proteins that are involved in a variety of cellular processes (Flower, 1996). A member of the lipocalin protein family, lipocalin 2 (LCN2) (24p3 (murine ortholog) or neutrophil gelatinase associated lipocalin (NGAL, human ortholog)) is a 25 kDa protein associated with 92 kDa gelatinase/MMP9 from human neutrophils (Kjeldsen et al., 1993) and produced by mammalian hosts to bind bacterial siderophore and sequester free iron. Microbes release siderophores to scavenge iron from the environment, and this is important for pathogenic bacteria which need to acquire iron from the mammalian host, where iron is tightly bound to proteins such as hemoglobin, transferrin, lactoferrin and ferritin. By binding siderophores, LCN2 competes with bacteria for iron and could be part of an innate immune response (Barasch and Mori, 2004). Upon encountering invading bacteria, Toll-like receptors on immune cells stimulate the transcription, translation and secretion of LCN2 which limits bacterial growth by sequestrating iron-laden siderophore. Mice deficient in LCN2 have increased susceptibility to bacterial infection (Flo et al., 2004). LCN2 is also involved in iron-binding in mammalian tissues such as the kidney (Schmidt-Ott et al., 2006, Schmidt-Ott et al., 2007, Yang et al., 2002, Yang et al., 2003) and could protect the organ from acute ischemic injury (Mishra et al., 2005, Mori et al., 2005). In addition, LCN2 is a serum biomarker and cytokine (adipokine) that is elevated in obesity, insulin resistance and hyperglycemia (Wang et al., 2007), and regulates inflammation in adipocytes and macrophages (Zhang et al., 2008).

Kainate (KA) is a potent agonist at the AMPA and kainate classes of glutamate receptors in the CNS (Berger et al., 1986). Intracerebroventricular (icv) injection of KA in rats induces neuronal loss in select regions of the brain, in particular the hippocampal formation (Nadler et al., 1978, Nadler et al., 1980) via excitotoxicity-mediated cell death (Wang et al., 2005). KA injections activate astrocytes and microglia (Jorgensen et al., 1993, Mitchell et al., 1993, Ravizza et al., 2005, Rizzi et al., 2003, Wang et al., 2005) and increase the expression of inflammatory cytokines in the lesioned areas (Ravizza et al., 2005, Rizzi et al., 2003). Increased iron level is detected in the KA-lesioned hippocampus by particle induced X-ray emission (PIXE) (Ong et al., 1999), histochemistry (Huang and Ong, 2005, Wang et al., 2002) and atomic absorption spectroscopy (Ong et al., 2006). Excess iron is a feature of many neurodegenerative diseases and has a deleterious effect on cells (Kell, 2010, Moos and Morgan, 2004). Iron accumulates around small microvessels in the brain during hypercholesterolemia (Ong and Halliwell, 2004, Ong et al., 2004). With age, iron levels increase in brain regions that are affected by Alzheimer’s disease and Parkinson’s disease. High concentrations of reactive iron can predispose to oxidative stress-induced neuronal injury, and iron accumulation might increase the toxicity of environmental or endogenous toxins (Zecca et al., 2004).

Despite the importance of LCN2 in iron homeostasis, relatively little is known about LCN2 in the brain. LCN2 is secreted by astrocytes and microglia as an autocrine mediator of reactive astrocytosis (Lee et al., 2009) and deramification of activated microglia in vitro (Lee et al., 2007), but little information is available on the normal brain distribution of LCN2 in vivo, and especially changes after neuronal injury. The present study was carried out to elucidate the expression of LCN2 in the normal brain and kainate-induced excitotoxicity model of neuronal injury.

Section snippets

Animals and kainate injections

Male Wistar rats weighing approximately 200 g each were anesthetized with intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). KA (1.2 μl of 1 mg/ml) was stereotaxically injected into the right lateral ventricle (coordinates: 1.0 mm caudal to bregma, 1.5 mm lateral to the midline, 4.5 mm from the surface of the cortex) using a microliter syringe. Experimental control rats were injected with 1.2 μl of normal saline instead of KA. All procedures involving animals were approved by the

Distribution of LCN2 mRNA and protein levels in different regions of the normal rat brain

Little is known about the distribution of LCN2 in the normal brain in vivo, and the mRNA expression of LCN2 was thus evaluated in different parts of the untreated rat brain, including the olfactory bulb, striatum, hippocampus, frontal cortex, somatosensory cortex, thalamus, hypothalamus, cerebellum and brainstem. The values are presented as fold difference relative to the lowest level among the different brain regions, i.e., the hippocampus. Highest level of LCN2 mRNA was found in the olfactory

Discussion

The present study was carried out to examine the distribution and expression of lipocalin 2 in normal rat brain and to study the changes in LCN2 expression after KA lesions. Distribution of LCN2 mRNA of the normal brain was first investigated using real-time RT-PCR. The olfactory bulb showed the highest level of LCN2 mRNA – 7-fold greater than the hippocampus, which had the lowest LCN2 mRNA basal expression. The thalamus, hypothalamus, cerebellum and brainstem showed 3- to 4-fold greater

Acknowledgment

This work is supported by a grant from the National Medical Research Council of Singapore.

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