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(Stroke. 1996;27:1682-1687.)
© 1996 American Heart Association, Inc.

Articles

Immediate Early Gene Expression in Response to Cerebral Ischemia

Friend or Foe?

Paul T. Akins, MD, PhD; Philip K. Liu, PhD Chung Y. Hsu, MD, PhD

the Cerebrovascular Disease Section, Department of Neurology, Washington University School of Medicine, St Louis, Mo (P.T.A., C.Y.H.); and the Division of Restorative Neurology and Human Neurobiology, Baylor College of Medicine, Houston, Tex (P.K.L.).

	Abstract

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
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 futher investivation. 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.

Key Words: apoptosis • cerebral ischemia • DNA damage • gene expression

	Introduction

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
Both focal and global cerebral ischemia produce immediate biochemical changes in cerebral tissue. Increases in the expression of genes can be detected within minutes of the onset of dense ischemia. The genes with the most rapid response fall mainly in the category of immediate early genes. The products of many of these genes form transcription factors that regulate a number of genes known as late response genes. In tissue surviving hours to days after ischemia, a multitude of additional genes are expressed.

The functional significance of gene expression in response to cerebral ischemia is under investigation. These changes may be either beneficial (friend) or detrimental (foe). Alterations in gene expression likely play a major role in the recovery process after cerebral ischemia. On the other hand, the induction of genes involved in apoptotic cell death triggered by ischemia may lead to further tissue injury. Future therapies targeting gene expression could be directed at genes that mediate either recovery or apoptosis.

	Immediate Early Genes

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
In response to a wide range of stimuli, mRNA for immediate early genes appears in cells within minutes. Cells can transcribe mRNA for immediate early genes in the presence of protein synthesis inhibitors, indicating that the stockpiles of the proteins required for their synthesis are maintained.¹ In fact, inhibition of protein synthesis can increase the expression of immediate early genes, because some immediate early gene products provide negative feedback by repressing their own transcription.¹ Immediate early genes were first identified in cells exposed to mitogens, and they were therefore implicated in the regulation of the cell cycle.² Since then, many immediate early genes have been identified, and knowledge of their function has expanded considerably (Table 1).

View this table: [in this window] [in a new window]   Table 1. Immediate Early Response Genes Induced by Cerebral Ischemia

In the central nervous system, a diversity of stimuli can induce immediate early genes. Under normal conditions, immediate early gene expression is generally low. When the stimulus is physiological, immediate early gene expression is increased in discrete brain regions functionally related to the stimulus. For example, water deprivation selectively increases expression of c-fos in the paraventricular nucleus that produces antidiuretic hormone.³ In contrast, pathophysiological conditions such as seizures⁴ and cerebral ischemia (see below) lead to induction of immediate early genes in wide regions of the central nervous system. While they appear in the regions of direct injury, they are also detected in areas outside of initial injury.

Several steps are involved in the induction of immediate early genes^{1 5} (Fig 1). A stimulus such as the release of glutamate serves as the first messenger. Next, biochemical changes in the cytoplasm of the cell such as elevated intracellular calcium levels or second messengers activate protein kinases, in particular protein kinase A (cAMP-dependent) or C (calcium-dependent). These kinases then phosphorylate DNA binding proteins such as the serum response element (SRE) or the calcium/cAMP response element (Ca/CRE). The DNA binding proteins recognize specific binding sites in the genomic DNA called response elements. The response elements are present in the promoter region of genes and regulate transcription of the respective gene. The signal transduction pathway for immediate early genes therefore involves convergence of multiple signals onto DNA binding proteins that regulate immediate early gene expression through response elements such as SRE or Ca/CRE.

View larger version (46K): [in this window] [in a new window]   Figure 1. A cascade of steps leads to induction of immediate early genes. The release of extracellular neurotransmitters such as glutamate activate second messenger systems such as cAMP and phosphoinositides. These second messenger systems regulate protein kinases that can modify DNA binding proteins. DNA binding proteins recognize specific binding sites in immediate early genes and regulate gene transcription. Many immediate early genes are themselves transcription factors. The late response genes that they regulate may be involved in either recovery or apoptosis. IP3 indicates inositol trisphosphate; DAG, diacylglycerol; AA, arachidonic acid metabolites; and G, G protein.

The prototype immediate early gene, c-fos, codes for a 380–amino acid protein known as the Fos protein (Fig 2). The fos gene was one of the first immediate early genes discovered. It was sequenced from the v-fos oncogene found in the Finkel-Biskis-Jinkins murine osteogenic sarcoma virus. Subsequently, the c-fos proto-oncogene was found in the genome of eukaryotes. The Fos protein contains several important structural features. A region of basic amino acids constitutes a DNA binding region. Nearby is a region containing leucine residues every 7 amino acids known as the leucine zipper. It forms an alpha helix with the leucines aligned on one side. Leucine zippers can align with other proteins containing this structure to form dimers. The dimers bind to a specific DNA region known as the AP-1 site,¹ which regulates the expression of a number of late effector genes.

View larger version (14K): [in this window] [in a new window]   Figure 2. Members of the fos and jun immediate early gene families form transcription factors through dimerization. The Fos and Jun proteins contain a leucine zipper structure that promotes the formation of dimers. The dimers recognize a specific DNA sequence called the AP-1 site present in many genes.

Because members of the fos and jun families contain the leucine zipper, many dimerization products could be formed in theory. However, these genes do exhibit some restraint.^{1 5} For example, the products of the genes c-fos, fos-B, and fra-1 form heterodimers with members of the jun family but do not form homodimers. Alternatively, the products of c-jun, jun-B, and jun-D will form both homodimers and heterodimers. The binding efficiency of dimers for the AP-1 site is affected by the subunit composition.⁶ The composition of the dimer may also determine whether the late effector gene is turned on or off.¹ Examples of genes known to contain AP-1 sites include neurotrophins, proenkephalin, glial fibrillary acidic protein, neuropeptide Y, vasoactive intestinal peptide, and tyrosine hydroxylase.⁷ Additional immediate early genes have been identified with other important structural elements and functions (Table 1). For example, the gene nur/77 is a member of the steroid receptor superfamily. The zif/268 protein contains a zinc finger that binds to a guanylate-rich DNA consensus sequence. The regulation of these genes may involve additional pathways.

In summary, the pathways leading to immediate early gene expression contain both convergent and divergent steps. A wide range of stimuli can converge to activate a limited number of second messenger systems. Through the activation of specific DNA binding proteins, the transcription of immediate early genes is regulated. Many immediate early gene products then regulate target genes. Conceptually, immediate early genes individually or in combination may control functionally related sets of late effector genes. The late effector genes may work in concert to accomplish a coordinated task. In the setting of cerebral ischemia, this could represent either the task of recovery or, alternatively, cell death.

	Immediate Early Gene Expression After Cerebral Ischemia

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
Cerebral ischemia is a potent stimulator of immediate early gene expression (Table 1). In animal models for global cerebral ischemia, either the forebrain or the whole brain is hypoperfused, yet discrete brain regions respond by inducing immediate early genes.^{8 9 10 11 12 13} With the use of the in situ hybridization technique, which anatomically localizes specific mRNA, increases in immediate early genes are detected predominantly in brain regions known to be most susceptible to ischemia.¹¹ For example, large increases in c-fos mRNA are present in the dentate granule cells, the CA1 and CA3 pyramidal neurons, the neocortex, and the Purkinje cells of the cerebellum after four-vessel occlusion in the rat.¹¹ Other immediate early genes including zif/268, c-jun, jun-B, and jun-D are also induced by global ischemia.¹²

Under the conditions of global cerebral ischemia, protein synthesis is reduced. Consequently, mRNA for immediate early genes may not be uniformly translated into the respective protein. Immunocytochemical studies with antibodies directed against the products of immediate early genes have addressed this question.¹² In general, the corresponding proteins are detected. An important exception occurs in the CA1 neurons. Immunoreactivity for the products of c-fos and zif/268 is not detected, despite large increases in their respective mRNA levels.¹² These neurons are highly vulnerable to ischemia.

The precise molecular mechanisms leading to immediate early gene expression are incompletely characterized. Global ischemia induces multiple second messenger systems presumably involved in the induction of immediate early genes. Expression of immediate early genes by ischemia may also reflect metabolic stress.^{14 15} N-Methyl-D-aspartate receptor antagonists reduce the expression of immediate early genes, suggesting that glutamate may be involved early in the signaling pathway.¹⁶

Focal cerebral ischemia also regulates immediate early gene expression.^{17 18 19 20 21 22 23} With mild focal ischemia, immediate early gene expression is limited to the ischemic zone. With more severe degrees of ischemia, immediate early gene expression is detected beyond the ischemic region. For example, with a rat stroke model of reversible focal ischemia, increases in c-jun, jun-B, jun-D, zif/268, c-fos, nur/77, and Krox 20 were detected within 1 hour of reperfusion.^{18 19} If the ischemia was increased from 30 to 90 minutes, the expression of immediate early genes extended beyond the ischemic zone to include the ipsilateral hippocampus. With a permanent middle cerebral artery (MCA) occlusion model in rats, c-fos, c-jun, and zif/268 expression was noted in ipsilateral cortical structures beyond the territory of the occluded vessel.²⁰ In another focal stroke model, c-fos and jun-B expression was detected in deeper structures, including the thalamus, the lateral and medial geniculate nuclei, and the substantia nigra.^{21 22} These remote effects bring to mind the results of in vivo cerebral blood flow and metabolism studies demonstrating diaschisis.¹⁴ Other mechanisms such as spreading cortical depression may be involved.

Successful translation of immediate early genes may occur after focal cerebral ischemia. Immunoreactivity for products of c-fos, fos-B, c-jun, jun-B, and jun-D was detected.^{23 24} As discussed earlier, dimerization of products of the fos and jun families leads to homodimers or heterodimers with AP-1 binding activity. Increases in AP-1 binding activity have been detected after focal cerebral ischemia.^{19 25 26} While mRNA for immediate early genes has been detected in both the core and penumbral regions of focal ischemia, detection of AP-1 binding was found only in the penumbral region.²⁶ This finding probably reflects the inhibition of protein synthesis that follows a dense ischemic insult.

Knowledge of the first and second messengers responsible for the increases in gene expression after focal cerebral ischemia is incomplete. Pretreatment with MK-801, a glutamate (N-methyl-D-aspartate) antagonist, reduces the expression of c-fos and jun-B in the ischemic regions after MCA occlusion.^{20 21} Under these conditions, immediate early gene expression still occurred in the substantia nigra,²¹ suggesting that other signals regulate immediate early gene expression in sites remote from the ischemic region.

The molecular mechanisms responsible for the induction of immediate early genes have been investigated. Nuclear run-on assays from ischemic tissue demonstrate increased transcription of c-jun, jun-B, c-fos, and Krox 20.^{18 19} This finding suggests that changes at the level of gene transcription are involved. Presumably, this effect is mediated by the activation of DNA binding proteins at the promoter regions of these genes. Indeed, increased binding activity to the cAMP response element (CRE), a response element present in the promoter region of c-fos, has been detected in nuclear extracts derived from ischemic brain tissue.^{10 25 26} It is unknown whether further regulation occurs at the posttranscription level.

	Additional Gene Expression After Cerebral Ischemia

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
In addition to immediate early genes, a multitude of other genes are induced by cerebral ischemia²⁷ (Table 2). The role of immediate early genes and other transcription factors in their induction requires further research. Heat shock proteins are rapidly induced by ischemia within the same time frame as immediate early genes and may be neuroprotective.¹⁵ Gene expression of trophic factors and their receptors is regulated by ischemia.^{28 29 30 31 32 33} Finklestein and colleagues^{28 29} were among the first to note an increase in the expression of growth factors such as basic fibroblast growth factor in the injured brain. Later, mRNA for nerve growth factor was found to be induced in the dentate gyrus within 24 hours of global ischemia.³⁰ Since an AP-1 site is present within the nerve growth factor gene, immediate early genes may be candidates for regulation of nerve growth factor expression.¹⁴ After focal cerebral ischemia, nerve growth factor and brain-derived neurotrophic factor mRNA can be detected within 4 hours of reperfusion.^{32 33} This expression first declines over 2 days and then increases in a biphasic manner during the next 3 weeks.

View this table: [in this window] [in a new window]   Table 2. Examples of Genes Regulated by Cerebral Ischemia

Expression of neurotransmitters and their receptors is also affected by cerebral ischemia. Neurotransmitter systems investigated include glutamate,^{34 35 36} -aminobutyric acid,^{37 38} and neuropeptides.³⁹ The mechanisms responsible for the possible induction of late response genes by immediate early genes are under investigation. Time-dependent changes in binding activities of transcription regulators, including those related to immediate early genes (AP-1, CREB, Sp-1, and NF-kB), have been detected.²⁶

	Apoptosis After Cerebral Ischemia

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
Evidence is accumulating that cerebral ischemia activates genes involved in both repair and destruction. Cerebral ischemia leads to cell death through two mechanisms: ischemic necrosis and apoptosis. Apoptotic cell death is an active pathway requiring macromolecular synthesis of products and may occur at a slower pace than ischemic necrosis.⁴⁰ Apoptosis is a physiological process in normal development and, under this circumstance, is referred to as programmed cell death. During apoptosis, DNA endonucleases are activated and cleave genomic DNA at regular intervals. DNA extracted from cells dying by this mechanism forms a pattern on DNA gels referred to as "DNA laddering." Histologically, cells that undergo apoptosis initially form clumps of DNA in the nucleus, blebbing of the plasma membrane, and shrinking of the cytoplasm. Minimal inflammation is seen. The genetics of this complex pathway are being elucidated in the Caenorhabditis elegans model.^{40 41} Many of these genes are highly conserved across species. Apoptosis has many stages, and it is likely that genes are involved that promote and retard each step.

The delayed neuronal injury that occurs in CA1 pyramidal neurons after global ischemia demonstrates many of the features associated with apoptosis.⁴² Cell death appears over several days with little inflammation. DNA laddering has been demonstrated.⁴³ Inhibition of protein synthesis with agents such as cycloheximide or anisomycin reduces cell loss,⁴⁴ demonstrating that the delayed death is an active process requiring the synthesis of new gene products.

Traditionally, infarction after focal cerebral ischemia has been attributed to ischemic necrosis. Recent experiments have provided evidence supporting a role for apoptotic death after focal cerebral ischemia.^{45 46} A transgenic mouse model has been established that overexpresses Bcl-2, a mitochondrial protein that inhibits apoptosis.⁴⁷ Compared with wild-type mice, these transgenic animals have smaller infarctions after comparable focal cerebral ischemic insults. Using an alternative strategy, Linnik et al⁴⁸ constructed a viral vector encoding for Bcl-2. They made cortical injections with the vector in wild-type rats before the onset of focal ischemia. Brain regions near the injections had greater survival than adjacent tissue, suggesting a neuroprotective effect of Bcl-2 by inhibiting apoptosis.

The duration and severity of the focal cerebral ischemic insult may determine whether tissue undergoes necrosis or apoptosis (Fig 3).⁴⁹ In a rat MCA occlusion model, a focal ischemic insult lasting 90 minutes produced an infarct whose volume was maximum 1 day later. This rapid destruction of tissue is consistent with a process dominated by ischemic necrosis. With focal ischemia lasting 30 minutes, no infarction was detected at 1 day of survival. However, a small infarct was detected at 3 days; by 2 weeks, the infarct was as large as the infarct produced by 90 minutes of ischemia.

View larger version (30K): [in this window] [in a new window]   Figure 3. Two pathways lead to cell death after cerebral ischemia. Dense ischemia causes rapid cell death, predominantly through necrosis. Mild to moderate ischemia may activate genes that regulate apoptotic cell death. Many genes are induced in brain tissue that survives the initial ischemic insult. They are predicted to play an important role in repair and recovery.

The delayed time course of infarction observed after mild focal ischemia is more consistent with apoptosis⁴⁹ than necrosis. Under these conditions, biochemical and histological features of apoptosis were seen, including DNA laddering and nuclear DNA condensation. The infarct volume after mild ischemia was reduced with protein synthesis inhibitors. Combination treatment with both a protein synthesis inhibitor and a glutamate antagonist acted synergistically to markedly reduce the infarct volume.⁵⁰ Cycloheximide also reduced infarct volume in a rat permanent MCA occlusion model.⁴⁵

The specific genes regulating apoptosis after cerebral ischemia are unknown. Under normal development in the nervous system, many neurons die by programmed cell death. This has been attributed to competition between neurons for a limited supply of trophic factors.⁴⁰ An experimental model has been developed based on the dependence of cultured sympathetic neuron survival on nerve growth factor. Removal of nerve growth factor from the growth medium results in death of approximately 50% of cells within 17 hours. Using a sensitive amplification method for mRNA, Estus et al⁵¹ characterized the time course of gene expression after the removal of nerve growth factor. In general, mRNA levels declined rapidly, including mRNA for the immediate early genes fra-1 and NF-kB and the anti-death gene Bcl-2. Two early phases of gene expression were noted. Within 5 hours of removal of nerve growth factor, increases in mRNA for c-jun, c-myb, and mkp-1 were detected. After 15 hours of trophic factor deprivation, a sharp increase in mRNA for the genes c-fos, fos-B, NGFI-A, and rhl appeared. Remarkably, intracellular injections of antibodies specific for c-jun and members of the c-fos family blocked apoptosis after nerve growth factor withdrawal.

Based on these results, the "Some Up, Some Down" model of programmed neuronal death or apoptosis was proposed.⁵¹ This model requires the coordinated induction of genes promoting apoptosis and repression of genes inhibiting it. As mentioned earlier, the subunit composition of the dimers formed by immediate early genes containing leucine zipper motifs determines the corresponding regulatory effects (induction versus repression) at the AP-1 site for the target genes.

Further research is needed to determine the role of immediate early genes in delayed cell death after cerebral ischemia in animal and tissue culture models. Anti-sense molecular probes designed to selectively block the translation of mRNA for a candidate gene may be useful to dissect out the specific role of candidate immediate early genes.^{7 14 25} Knockout transgenic animals are an alternative strategy. These experiments may be complicated by the notion that multiple genes are involved in both the activation and the termination of apoptotic cell death after cerebral ischemia.

	Recovery After Cerebral Ischemia

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
The recovery process after focal infarction is complex and probably regulated by a host of genes. Recovery after a stroke occurs in stages characterized by different rates of improvement and patterns of motor recovery. Altered expression of neurotrophic factors and neurotransmitter systems is predicted to be involved in this process. While selected growth factors appear to be neuroprotective after focal cerebral ischemia,⁵² it is of concern that some neurotrophins such as brain-derived neurotrophic factor exacerbated hypoxic-ischemic injury in cultured cortical neurons.⁵³

Delayed complications of strokes are also likely mediated by altered gene expression. Examples include spasticity, dystonia, neurogenic bladder syndromes, seizures, and poststroke depression. Additional knowledge of the mechanisms responsible for plasticity after cerebral ischemia may lead to new treatments designed to increase the rate and extent of stroke recovery. Ischemia can damage DNA directly.^{41 54} DNA lesions caused by ischemia may arise from the production of reactive oxygen species in close proximity to DNA. It is interesting that DNA repair by nucleotide excision is more active in the transcribed strand than in the nontranscribed strand.^{41 55} The functional significance is unclear at this time, but recovery may be impeded if genes important for repair and neuronal plasticity are damaged.

	Conclusion

Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
Cerebral ischemia is a potent stimulus for gene regulation. Rapid changes occur in a class of genes known as immediate early genes. Many immediate early genes are transcription factors that regulate a host of target genes. A large number of additional genes are regulated by cerebral ischemia. The functional significance of early and delayed gene regulation by ischemia requires further research. Many of these genes are likely neuroprotective and important for recovery after cerebral ischemia.

Evidence is accumulating that ischemia produces tissue injury through the combined pathways of ischemic necrosis and apoptosis. Apoptotic cell death is genetically regulated. Additional research is needed to identify genes involved in apoptosis after cerebral ischemia. Immediate early genes are strong candidates, since they have been implicated in neuronal apoptosis after the withdrawal of trophic factors. Agents designed to block apoptotic cell death may offer additional approaches to the treatment of acute ischemic stroke. Neurorehabilitation after cerebral ischemia is a gradual process requiring extensive neuronal remodeling. Identification of the genes involved and their function during rehabilitation may suggest novel strategies to improve clinical outcome after ischemic strokes.

	Acknowledgments

 
Research described in this review was supported in part by the Vivian L. Smith Foundation for Restorative Neurology, the American Heart Association (94012700 to Dr Liu), the National Institutes of Health (NS25545, NS28995, and NS32636 to Dr Hsu; NS34810 to Dr Liu), and the Office of Naval Research (C4114503-01 to Dr Hsu).

	Footnotes

 
Reprint requests to Chung Y. Hsu, MD, PhD, Cerebrovascular Disease Section, Department of Neurology, Washington University School of Medicine, Box 8111, 660 S Euclid Ave, St Louis, MO 63110. E-mail: hsuc@neuro.wustl.edu.

Received April 24, 1996; revision received June 3, 1996; accepted June 5, 1996.

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Top Abstract Introduction Immediate Early Genes Immediate Early Gene Expression... Additional Gene Expression After... Apoptosis After Cerebral... Recovery After Cerebral Ischemia Conclusion References

 
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