Philip K. Liu

Immediate Early Gene Expression in Response to Cerebral Ischemia: Friend or Foe?

BACKGROUND Cerebral ischemia is a potent modulator of gene expression. Immediate early genes undergo rapid induction after both global and focal cerebral ischemia. Many immediate early genes code for transcription factors. Additional genes, including those encoding for neurotrophic factors and neurotransmitter systems, are induced in a delayed fashion after cerebral ischemia. The functional significance of early and late gene regulation after cerebral ischemia requires further investigation. These changes may be beneficial (friend) or detrimental (foe). Many of the genes are likely neuroprotective and important for recovery, but others may be involved in ischemic cell death mediated by apoptosis. SUMMARY OF REVIEW We review evidence that supports the hypothesis that cell death after cerebral ischemia occurs through the dual pathways of ischemic necrosis and apoptosis. CONCLUSIONS Gene regulation, including immediate early genes, is required for programmed neuronal death after trophic factor deprivation and is predicted to be involved in apoptosis triggered by cerebral ischemia. Novel therapies following cerebral ischemia may be directed at genes mediating either recovery or apoptosis.

Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long‐Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30–90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N‐glycosylase (Fpg protein)‐sensitive sites (FPGSS), as labeled in situ using digoxigenin‐dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8‐hy‐droxy‐2′‐deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS‐positive cells in the cortex from the sham‐operated control group was 3 ± 3 (mean ± sd per mm2). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS‐positive cells were significantly increased by 200‐fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8‐hydroxy‐guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 μg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham‐operated control group. We detected few FPGSS of oh8dG‐positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked‐end labeling (TUNEL)‐positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.—Cui, J., Holmes, E. H., Greene, T. G., Liu, P. K. Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.

Up-regulation of base excision repair activity for 8-hydroxy-2'-deoxyguanosine in the mouse brain after forebrain ischemia-reperfusion.

The repair enzyme 8‐oxoguanine glycosylase/apyrimidinic/apurinic lyase (OGG) removes 8‐hydroxy‐2′‐deoxyguanosine (oh8dG) in human cells. Our goal was to examine oh8dG‐removing activity in the cell nuclei of male C57BL/6 mouse brains treated with either forebrain ischemia‐reperfusion (FbIR) or sham operations. We found that the OGG activity in nuclear extracts, under the condition in which other nucleases did not destroy the oligodeoxynucleotide duplex, excised oh8dG with the greatest efficiency on the oligodeoxynucleotide duplex containing oh8dG/dC and with less efficiency on the heteroduplex containing oh8dG/dT, oh8dG/dG, or oh8dG/dA. This specificity was the same as for the recombinant type 1 OGG (OGG1) of humans. We observed that the OGG1 peptide and its activity in the mouse brain were significantly increased after 90 min of ischemia and 20‐30 min of reperfusion. The increase in the protein level and in the activity of brain OGG1 correlated positively with the elevation of FbIR‐induced DNA lesions in an indicator gene (the c‐fos gene) of the brain. The data suggest a possibility that the OGG1 protein may excise oh8dG in the mouse brain and that the activity of OGG1 may have a functional role in reducing oxidative gene damage in the brain after FbIR.

Increased expression of c-fos mRNA and AP-1 transcription factors after cortical impact injury in rats

Levels of c-fos mRNA and AP-1 transcription factors co-expression were measured in a controlled lateral cortical impact model of traumatic brain injury (TBI) in rats. Ipsilateral cerebral cortex and bilateral hippocampal c-fos mRNA increases were revealed by in situ hybridization after lateral cortical impact injury. Based on regional in situ hybridization data, we employed semi-quantitative RT-PCR methods to study the temporal profile of changes in the ipsilateral cortex at the site of injury. We found that TBI produces transient increases of c-fos mRNA expression in the ipsilateral cerebral cortex at 5 min postinjury, which peaks at 1 h postinjury and subsides by 1 day postinjury. Gel shift nuclear protein binding assays showed that AP-1 transcription factor binding was robustly increased in injured cerebral cortex at 1 h, 3 h, 5 h and 1 day after injury. These data indicate that TBI can produce significant increases in c-fos expression and subsequent upregulation of the AP-1 transcription factors. Thus, AP-1 transcription factors modulation of downstream gene expression may be an important component of pathophysiological responses to TBI.

Isolation and characterization of a UV-sensitive hypermutable aphidicolin-resistant Chinese hamster cell line

Aphidicolin is a specific inhibitor of DNA polymerase α and blocks DNA synthesis in vivo. The inhibition of purified α-polymerase has been shown to be competitive with dCTP but not with the other three deoxynucleoside triphosphates (dNTPs). In order to study the various roles that the α-polymerase might play in DNA replication and/or repair, we have attempted to isolate Chinese hamster V79 cells that are resistant to aphidicolin. Four resistant mutants were isolated from BrdU-black light- and UV-mutagenized cells. None of the mutants isolated contains an α-polymerase that is resistant, in crude extract measurements, to aphidicolin. Three mutants isolated, however, were found to be resistant to araC. Two mutants tested were found to be sensitive to cytidine and have elevated levels of dCTP or all 4 dNTPs. These results indicate that they are nucleotide pool mutants instead of α-polymerase mutants. One mutant, aphr-4, is characterized by the following: (1) high level of dCTP; (2) thymidine (or CdR, UdR, auxotrophic; (3) sensitive to thymidine (and AdR, GdR); (4) slow-growing; (5) cytidine sensitive; (6) UV sensitive and hypermutable at the ouabain-resistant locus; and (7) a ninefold increase in frequency of chromatid gaps and breaks when cells are exposed to BrdU- containing medium. Revertants of aphr-4 which are partially aphidicolin-resistant and retain the first three characteristics listed above, but not the others, have been isolated. The appearance of this type of revertant indicates that either aphr- 4 or its “revertant” is a double mutant.

Oxidative Damage to the c-fos Gene and Reduction of Its Transcription After Focal Cerebral Ischemia

Abstract : We investigated oxidative damage to the c‐fos gene and to its transcription in the brain of Long‐Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model. We observed a significant (p < 0.001) increase in the immunoreactivity to 8‐hydroxy‐2′‐guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0‐30 min of reperfusion in all 27 animals treated with 15‐90 min of ischemia. Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3‐bromo‐7‐nitroindazole (60 mg/kg, i.p.), abolished the majority but not all of the oh8G/oh8dG immunoreactivity. Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted. This observation was further supported by decreased levels of mRNA transcripts of the c‐fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization. The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c‐fos gene, was increased 13‐fold (p < 0.01). Our results suggest that inhibiting nNOS partially attenuates FCIR‐induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain.

Inhaled Nitric Oxide Improves Outcomes After Successful Cardiopulmonary Resuscitation in Mice

Background— Sudden cardiac arrest (CA) is a leading cause of death worldwide. Breathing nitric oxide (NO) reduces ischemia/reperfusion injury in animal models and in patients. The objective of this study was to learn whether inhaled NO improves outcomes after CA and cardiopulmonary resuscitation (CPR). Methods and Results— Adult male mice were subjected to potassium-induced CA for 7.5 minutes whereupon CPR was performed with chest compression and mechanical ventilation. One hour after CPR, mice were extubated and breathed air alone or air supplemented with 40 ppm NO for 23 hours. Mice that were subjected to CA/CPR and breathed air exhibited a poor 10-day survival rate (4 of 13), depressed neurological and left ventricular function, and increased caspase-3 activation and inflammatory cytokine induction in the brain. Magnetic resonance imaging revealed brain regions with marked water diffusion abnormality 24 hours after CA/CPR in mice that breathed air. Breathing air supplemented with NO for 23 hours starting 1 hour after CPR attenuated neurological and left ventricular dysfunction 4 days after CA/CPR and markedly improved 10-day survival rate (11 of 13; P=0.003 versus mice breathing air). The protective effects of inhaled NO on the outcome after CA/CPR were associated with reduced water diffusion abnormality, caspase-3 activation, and cytokine induction in the brain and increased serum nitrate/nitrite levels. Deficiency of the &agr;1 subunit of soluble guanylate cyclase, a primary target of NO, abrogated the ability of inhaled NO to improve outcomes after CA/CPR. Conclusions— These results suggest that NO inhalation after CA and successful CPR improves outcome via soluble guanylate cyclase–dependent mechanisms.

Ischemic injury and faulty gene transcripts in the brain.

The brain has the highest metabolic rate of all organs and depends predominantly on oxidative metabolism as a source of energy. Oxidative metabolism generates reactive oxygen species, which can damage all cellular components, including protein, lipids and nucleic acids. The processes of DNA repair normally remove spontaneous gene damage with few errors. However, cerebral ischemia followed by reperfusion leads to elevated oxidative stress and damage to genes in brain tissue despite a functional mechanism of DNA repair. These critical events occur at the same time as the expression of immediate early genes, the products of which trans-activate late effector genes that are important for sustaining neuronal viability. These findings open the possibility of applying genetic tools to identify molecular mechanisms of gene repair and to derive new therapies for stroke and brain injury.

Imaging Cerebral Gene Transcripts in Live Animals

To circumvent the limitations of using postmortem brain in molecular assays, we used avidin–biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15–20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and β-actin mRNA (SPION-cfos and SPION-βactin, respectively) (14–22 nm). The Stern–Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 × 106/m. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (MRI) to monitor gene transcription, and demonstrated that these complexes at 40 μg of Fe per kilogram of body weight were retained at least 1 d after intracerebroventricular infusion into the left ventricle of C57Black6 mice. SPION retention measured by MRI as T2* or R2* maps (R2* = 1/T2*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T2* signal in the right cortex and striatum; retention of SPION-cfos and SPION-βactin positively correlated with c-fos and β-actin mRNA maps obtained from in situ hybridization. Histological examination showed that intracellular iron oxide and FITC-sODN correlated positively with in vivo MR signal reduction. Furthermore, in animals that were administered SPION-cfos and amphetamine (4 mg/kg, i.p.), retention was significantly elevated in the nucleus accumbens, striatum, and medial prefrontal cortex of the forebrain. Control groups that received SPION-cfos and saline or that received a SPION conjugate with a random-sequence probe and amphetamine showed no retention. These results demonstrated that SPION-sODN conjugates can detect active transcriptions of specific mRNA species in living animals with MRI.

The association between neuronal nitric oxide synthase and neuronal sensitivity in the brain after brain injury.

Abstract: Injury to the central nervous system is the leading cause of disability in the United States. Neuronal death is one of the causes of disability. Among patients who survive this type of injury, various degrees of recovery in brain function are observed. The molecular basis of functional recovery is poorly understood. Clinical observations and research using experimental injury models have implicated several metabolites in the cascade of events that lead to neuronal degeneration. The levels of intracellular ATP (energy source) and pH are decreased, whereas levels of extracellular glutamate, intracellular calcium ions, and oxidative damage to RNA/DNA, protein, and lipid are increased. These initiating events can be associated with energy failure and mitochondrial dysfunction, resulting in functional or structural brain damage. The injured brain is known to express immediate early genes. Recent studies show that reactive oxygen species (ROS) cause lesions in genes from which mRNA is transcribed as part of the endogenous neuroprotective response. Although degenerating proteins and lipids may contribute to necrosis significantly after severe injury, abnormalities in genetic material, if not repaired, disturb cellular function at every level by affecting replication, transcription, and translation. These lesions include abnormal nucleic acids, known as oxidative lesions of DNA (ODLs) or of RNA (ORLs). In this review, we focus on our current understanding of the various effects of neuronal nitric oxide synthase on the formation of modified bases in DNA and RNA that are induced in the brain after injury, and how ODLs and ORLs affect cell function.