Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin-2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose-dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke-reperfusion injury.

ATR [ataxia-telangiectasia-mutated (ATM)-and Rad3-related] is a protein kinase required for both DNA damage-induced cell cycle checkpoint responses and the DNA replication checkpoint that prevents mitosis before the completion of DNA synthesis. Although ATM and ATR kinases share many substrates, the different phenotypes of ATM-and ATR-deficient mice indicate that these kinases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on Ser 635 and Ser 645 in vitro. In undamaged synchronized human cells, these two sites were phosphorylated in late G 1, S, and G2͞M, but not in early-mid G1. Treatment of cells with genotoxic stress induced phosphorylation of hRad17 in cells in early-mid G 1. Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues were phosphorylated in ionizing radiation (IR)-treated ATM-deficient human cell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-deficient tissues, but induction of Ser 645 was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR-induced activation of the G 1͞S checkpoint in MCF-7 cells. These results suggest ATR and hRad17 are essential components of a DNA damage response pathway in mammalian cells. C ell cycle checkpoints activated by stalled replication forks and DNA damage protect genomic integrity by preventing damaged DNA from being replicated and passed on to new daughter cells (1-5). In Schizosaccharomyces pombe (Sp), conserved checkpoint Rad proteins, including Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1, are required for activation of checkpoint signaling pathways in response to stalled replication forks and DNA damage. Inactivation of any one of the checkpoint rad genes abolishes phosphorylation and activation of two downstream kinases, SpChk1 and SpCds1 (6, 7), resulting in defective activation of checkpoints. Human homologues of all of the Sp checkpoint rad genes have been identified, except rad26.The Sprad3 ϩ gene, the Saccharomyces cerevisiae (Sc) MEC1 gene, and the human ATM (ataxia-telangiectasia-mutated) and ATR (ATM-and Rad3-related) genes encode related protein kinases (8, 9). ATR and ATM are involved in the replication and DNA damage-induced checkpoints (10-14). Although ATM has been extensively studied, it has been difficult to ascertain ATR function because ATR-deficient cells and embryos are not viable (15,16). ATM deficiency results in hypersensitivity to ionizing irradiation (IR) in humans and mice (17,18). Similarly, ATRdeficient blastocysts have increased sensitivity to IR that correlates with chromosomal fragmentation (15). By using cells overexpressing kinase-inactive ATR (ATR Ki ) under the regulation of doxycycline, an elevated cellular sensitivity to DNA damage, a defective cell cycle response, and a significant loss of cell viability were observed (13,19). Cellular substrates of ATM͞ATR include p53 (20-23) and BRCA1 (24, 25), but substra...

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