Matrix-derived inflammatory mediator N-acetyl proline-glycine-proline is neurotoxic and upregulated in brain after ischemic stroke.

BACKGROUND: N-acetyl proline-glycine-proline (ac-PGP) is a matrix-derived chemokine produced through the proteolytic destruction of collagen by matrix metalloproteinases (MMPs). While upregulation and activation of MMPs and concomitant degradation of the extracellular matrix are known to be associated with neurological injury in ischemic stroke, the production of ac-PGP in stroke brain and its effects on neurons have not been investigated. FINDINGS: We examined the effects of ac-PGP on primary cortical neurons and found that it binds neuronal CXCR2 receptors, activates extracellular signal-regulated kinase 1/2 (ERK1/2), and induces apoptosis associated with caspase-3 cleavage in a dose-dependent manner. After transient ischemic stroke in rats, ac-PGP was significantly upregulated in infarcted brain tissue. CONCLUSIONS: The production of ac-PGP in brain in ischemia/reperfusion injury and its propensity to induce apoptosis in neurons may link MMP-mediated destruction of the extracellular matrix and opening of the blood-brain barrier to progressive neurodegeneration associated with the initiation and propagation of inflammation. Ac-PGP may be a novel neurotoxic inflammatory mediator involved in sustained inflammation and neurodegeneration in stroke and other neurological disorders associated with activation of MMPs.

Identification of isoxsuprine hydrochloride as a neuroprotectant in ischemic stroke through cell-based high-throughput screening.

Stroke is a leading cause of death and disability and treatment options are limited. A promising approach to accelerate the development of new therapeutics is the use of high-throughput screening of chemical libraries. Using a cell-based high-throughput oxygen-glucose deprivation (OGD) model, we evaluated 1,200 small molecules for repurposed application in stroke therapy. Isoxsuprine hydrochloride was identified as a potent neuroprotective compound in primary neurons exposed to OGD. Isoxsuprine, a ß2-adrenergic agonist and NR2B subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonist, demonstrated no loss of efficacy when administered up to an hour after reoxygenation in an in vitro stroke model. In an animal model of transient focal ischemia, isoxsuprine significantly reduced infarct volume compared to vehicle (137 ± 18 mm3 versus 279 ± 25 mm3, p < 0.001). Isoxsuprine, a peripheral vasodilator, was FDA approved for the treatment of cerebrovascular insufficiency and peripheral vascular disease. Our demonstration of the significant and novel neuroprotective action of isoxsuprine hydrochloride in an in vivo stroke model and its history of human use suggest that isoxsuprine may be an ideal candidate for further investigation as a potential stroke therapeutic.

Transient middle cerebral artery occlusion with complete reperfusion in spontaneously hypertensive rats.

Middle cerebral artery occlusion (MCAO) by the intraluminal suture method is widely used to model ischemic stroke in rats. Current methods include transection or ligation of the external carotid or common carotid artery and thus result in partial restoration of perfusion after transient MCAO. Since incomplete reperfusion may influence recovery and thus confound studies of the impact of neuroprotective compounds and therapies on outcomes after stroke, we have devised a novel method to induce transient MCAO with complete reperfusion. Advantages of the method include: MCAO is achieved through insertion of an intraluminal suture into the internal carotid artery through the common carotid artery.At the end of the occlusion period, the suture is withdrawn and the incision in the common carotid artery is closed with cyanoacrylate tissue adhesive and complete reperfusion is established.No residual subcutaneous sutures remain during recovery.Vasculature is restored to the preoperative state.

Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery.

In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood-brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.

Genetic polymorphisms of multiple DNA repair pathways impact age at diagnosis and TP53 mutations in breast cancer.

Defective DNA repair may contribute to early age and late stage at time of diagnosis and mutations in critical tumor suppressor genes, such as TP53 in breast cancer. Using DNA samples from 436 breast cancer cases (374 Caucasians and 62 African-Americans), we tested these associations with 18 non-synonymous single-nucleotide polymorphisms (nsSNPs) in four DNA repair pathways: (i) base excision repair: ADPRT V762A, APE1 D148E, XRCC1 R194W/R280H/R399Q and POLD1 R119H; (ii) double-strand break repair: NBS1 E185Q and XRCC3 T241M; (iii) mismatch repair: MLH1 I219V, MSH3 R940Q/T1036A and MSH6 G39E and (iv) nucleotide excision repair: ERCC2 D312N/K751Q, ERCC4 R415Q, ERCC5 D1104H and XPC A499V/K939Q. Younger age at diagnosis (<50) was associated with ERCC2 312 DN/NN genotypes [odds ratio (OR) = 1.76; 95% confidence interval (CI) = 1.10, 2.81] and NBS1 185 QQ genotype (OR = 3.09; 95% CI = 1.47, 6.49). The XPC 939 QQ genotype was associated with TP53 mutations (OR = 5.80; 95% CI = 2.23, 15.09). There was a significant trend associating younger age at diagnosis (<50) with increasing numbers of risk genotypes for ERCC2 312 DN/NN, MSH6 39 EE and NBS1 185 QQ (P(trend) < 0.001). A similar significant trend was also observed associating TP53 mutations with increasing numbers of risk genotypes for XRCC1 399 QQ, XPC 939 QQ, ERCC4 415 QQ and XPC 499 AA (P(trend) < 0.001). Our pilot data suggest that nsSNPs of multiple DNA repair pathways are associated with younger age at diagnosis and TP53 mutations in breast cancer and larger studies are warranted to further evaluate these associations.

Multiple roles of metalloproteinases in neurological disorders.

Once thought to mainly act in brain to remodel the extracellular matrix, the family of metalloproteinases is important in many normal and pathological processes in the nervous system. Matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) are the two major families of metalloproteinases in the brain. MMPs are comprised of several related enzymes that act on extracellular molecules. Normally, they are important in angiogenesis and neurogenesis in development. In neuroinflammatory illnesses, they disrupt the basal lamina and tight junction proteins to open the blood-brain barrier (BBB). ADAMs are important in neuroinflammation through activation of tumor necrosis factor-α (TNF-α) and their action as secretases that modulate the action of receptors on the cell surface. Four tissue inhibitors of metalloproteinases (TIMPs) are the main inhibitors of the MMPs and ADAMs. Recently, MMPs were found to affect DNA repair processes by an unexpected intranuclear action. MMPs and ADAMs have been implicated in the pathophysiology of neurodegenerative diseases such as Alzheimer's disease and vascular cognitive impairment. Growing literature on the functions of MMPs and ADAMs in the central nervous system is opening up new and exciting areas of research that may lead to novel approaches to treatment of neurological diseases.

Validation of the cell cycle G(2) delay assay in assessing ionizing radiation sensitivity and breast cancer risk.

Genetic variations in cell cycle checkpoints and DNA repair genes are associated with prolonged cell cycle G(2) delay following ionizing radiation (IR) treatment and breast cancer risk. However, different studies reported conflicting results examining the association between post-IR cell cycle delay and breast cancer risk utilizing four different parameters: cell cycle G(2) delay index, %G(2)-M, G(2)/G(0)-G(1), and (G(2)/G(0)-G(1))/S. Therefore, we evaluated whether different parameters may influence study results using a data set from 118 breast cancer cases and 225 controls as well as lymphoblastoid and breast cancer cell lines with different genetic defects. Our results suggest that cell cycle G(2) delay index may serve as the best parameter in assessing breast cancer risk, genetic regulation of IR-sensitivity, and mutations of ataxia telangiectasia mutated (ATM) and TP53. Cell cycle delay in 21 lymphoblastoid cell lines derived from BRCA1 mutation carriers was not different from that in controls. We also showed that IR-induced DNA-damage signaling, as measured by phosphorylation of H2AX on serine 139 (γ-H2AX) was inversely associated with cell cycle G(2) delay index. In summary, the cellular responses to IR are extremely complex; mutations or genetic variations in DNA damage signaling, cell cycle checkpoints, and DNA repair contribute to cell cycle G(2) delay and breast cancer risk. The cell cycle G(2) delay assay characterized in this study may help identify subpopulations with elevated risk of breast cancer or susceptibility to adverse effects in normal tissue following radiotherapy.

OGG1 is degraded by calpain following oxidative stress and cisplatin exposure.

Deficient repair activity for 8-hydroxy-2'-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer's disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca(2+)-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (proline, glutamic acid, serine, threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion, and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.

A novel R229Q OGG1 polymorphism results in a thermolabile enzyme that sensitizes KG-1 leukemia cells to DNA damaging agents.

BACKGROUND: Mutations and polymorphisms of OGG1, the major mammalian 8-oxoguanine repair activity, are associated with increased risk for several cancers. Decreased 8-oxoguanine repair capacity due to variant forms of the OGG1 gene is a common feature of numerous cancer cell lines. One such cell line, human KG-1 leukemia cells, has previously been demonstrated to be deficient in the excision of 8-oxoguanine from oxidatively damaged DNA. KG-1 cells have a homozygous R229Q amino acid substitution in OGG1 that has been presumed to alter the function of OGG1 and result in elevated levels of genomic 8-oxoG and hypersensitivity to 8-hydroxydeoxyguanosine nucleoside and ionizing radiation observed in KG-1 cells. METHODS: We characterized the enzymatic activity of R229Q OGG1 and the effect of the enzyme on cell survival following treatment with DNA damaging agents. RESULTS: R229Q OGG1 had activity similar to the wild-type enzyme, yet was easily heat inactivated at physiological temperature. R229Q OGG1 expressed in human cells had significantly lower activity than wild-type OGG1 and was also highly thermolabile. Expression of R229Q OGG1 sensitized KG-1 cells to killing by menadione and 8-hydroxydeoxyguanosine, but not ionizing radiation. CONCLUSIONS: These results suggest that decreased 8-oxoguanine repair in KG-1 is due to thermolability of R229Q OGG1 and that the enzyme variant increases cellular susceptibility to killing resulting from oxidative DNA damage. The R229Q OGG1 variant is a validated polymorphism prevalent in world populations and not an isolated mutation in KG-1 cells, thus the R229Q OGG1 allele may be a novel marker for cancer susceptibility.

Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase.

Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base lesion produced by reactive oxygen species (ROS). A frequently occurring OGG1 polymorphism in human populations results in the substitution of serine 326 for cysteine (S326C). The 326 C/C genotype is linked to numerous cancers, although the mechanism of carcinogenesis associated with the variant is unclear. We performed detailed enzymatic studies of polymorphic OGG1 and found functional defects in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the wild-type enzyme, depending upon the base opposite the lesion. Binding experiments showed that the polymorphic OGG1 binds DNA damage with significantly less affinity than the wild-type enzyme. Remarkably, gel shift, chemical cross-linking and gel filtration experiments showed that S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was also found to have reduced activity and a dimeric conformation. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack of stimulation by AP-endonuclease 1 (APE1) and anomalous DNA binding conformation of S326C OGG1 may contribute to its linkage to cancer incidence.

Choreography of oxidative damage repair in mammalian genomes.

The lesions induced by reactive oxygen species in both nuclear and mitochondrial genomes include altered bases, abasic (AP) sites, and single-strand breaks, all repaired primarily via the base excision repair (BER) pathway. Although the basic BER process (consisting of five sequential steps) could be reconstituted in vitro with only four enzymes, it is now evident that repair of oxidative damage, at least in mammalian cell nuclei, is more complex, and involves a number of additional proteins, including transcription- and replication-associated factors. These proteins may be required in sequential repair steps in concert with other cellular changes, starting with nuclear targeting of the early repair enzymes in response to oxidative stress, facilitation of lesion recognition, and access by chromatin unfolding via histone acetylation, and formation of metastable complexes of repair enzymes and other accessory proteins. Distinct, specific subclasses of protein complexes may be formed for repair of oxidative lesions in the nucleus in transcribed vs. nontranscribed sequences in chromatin, in quiescent vs. cycling cells, and in nascent vs. parental DNA strands in replicating cells. Characterizing the proteins for each repair subpathway, their signaling-dependent modifications and interactions in the nuclear as well as mitochondrial repair complexes, will be a major focus of future research in oxidative damage repair.