Journal of the Neurological Sciences 179 (2000) 1–33www.elsevier.com/ locate / jns

Review article

Brain ischemia and reperfusion: molecular mechanisms of neuronal injury

a,b , a a,b a*Blaine C. White , Jonathon M. Sullivan , Donald J. DeGracia , Brian J. O’Neil ,e d c a´Robert W. Neumar , Lawrence I. Grossman , Jose A. Rafols , Gary S. Krause

aDepartment of Emergency Medicine, Wayne State University School of Medicine, Detroit, MI, USAbDepartment of Physiology, Wayne State University School of Medicine, Detroit, MI, USA

cDepartment of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI, USAdCenter for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA

eDepartment of Emergency Medicine, University of Pennsylvania, Philadelphia, PA, USA

Received 8 May 2000; received in revised form 10 July 2000; accepted 12 July 2000

Abstract

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity inpartitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence andreduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization,which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-

21regulated calcium channels. This allows a large increase in cytosolic Ca associated with activation of m-calpain, calcineurin, andphospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of

21Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca from the ER lumen. A kinase that shuts offtranslation initiation by phosphorylating the a-subunit of eukaryotic initiation factor-2 (eIF2a) is activated either by adenosine degradation

21products or depletion of ER lumenal Ca . Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excessoxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result inperoxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damagethe plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2a phosphorylation that in SVNs is prolonged andassociated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drugadministration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associatedwith down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours ofreperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membranedamage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms thatsubstantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept ofsingle drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannotbe effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growthfactors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3)inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional andhistopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug. 2000Elsevier Science B.V. All rights reserved.

Keywords: Brain ischemia; Lipid peroxidation; Translation initiation; Apoptosis; Growth factors; Signal transduction

*Corresponding author. Department of Emergency Medicine, 6G University Health Care Center, 4201 St. Antoine, Detroit, MI 48201, USA. Tel.:11-313-577-5738; fax: 11-313-577-4131.

E-mail address: bwhite@med.wayne.edu (B.C. White).

0022-510X/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-510X( 00 )00386-5

2 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

1. Introduction sufficiently understand the mechanisms involved in neuro-nal injury and repair to design clinically effective therapy.

Stroke is the leading cause of serious, long-term dis- There are as yet no clinically effective therapeutic proto-ability in the United States [1], with about 600,000 people cols for amelioration of brain damage by ischemia andsuffering a new or recurrent stroke each year. Three reperfusion. In the 1980s clinical trials of barbiturate-million Americans are currently permanently disabled induced coma [3] or calcium antagonists [4] failed tobecause of ischemic stroke, and 31% of stroke survivors reduce neurologic damage caused by cardiac arrest andneed help caring for themselves, 20% need help walking, resuscitation. More recently, both the radical scavenger71% have an impaired vocational capacity when examined tirilazad [5] and the glutamate receptor antagonist selfotelan average of 7 years later, and 16% have to be in- [6] have been found ineffective in clinical treatment ofstitutionalized. The direct and indirect cost of stroke in stroke. Thus the theoretical syntheses that led to these trials1998 is estimated at $43.3 billion [1]. In addition to this, were inadequate. Here we will examine new informationcardiopulmonary resuscitation for victims of cardiac arrest, regarding neuronal injury (Fig. 1) and repair, indicatingboth within and outside of the hospital, succeeds in that:restoring spontaneous circulation in about 70,000 patientsa year in the United States. At least 60% of these patientssubsequently die in the hospital as a result of extensive 1. Many phenomena observed during brain ischemia andbrain damage; only 3–10% of resuscitated patients are reperfusion can be accounted for by damage tofinally able to resume their former lifestyles [2]. Thus, membrane lipids, specifically by lipolysis during is-brain injury by transient complete global brain ischemia chemia and by radical-mediated peroxidation of poly-(cardiac arrest) and regional incomplete brain ischemia unsaturated fatty acids (PUFAs) during reperfusion.(ischemic stroke) afflicts a very large number of patients 2. Protein synthesis in vulnerable brain neurons is rapidlywith death or permanent disability. and persistently inhibited at the level of translation

In order to reduce this neurologic morbidity, we must initiation during post-ischemic reperfusion.

21Fig. 1. Ischemic energy depletion leads to increased neuronal cytosolic [Ca ] through pump failure and depolarization- and neurotransmitter-opened21channels. Enzymes activated by cytosolic Ca overload include the protease m-calpain, the phosphatase calcineurin, and phospholipase A2; phospholipase

C is activated directly by depolarization. Cytoskeletal proteins and eIF4G (docks capped-mRNAs for translation) are degraded by m-calpain.Dephosphorylation by calcineurin activates NOS and releases Bad from sequestering proteins. Phospholipases liberate free fatty acids, particularly

21arachidonate, from membranes. Arachidonate causes depletion of ER Ca , inducing the ER unfolded protein response that includes activation of a eIF2a

kinase. Reperfusion allows superoxide generation during arachidonate metabolism and NOS production of NO, which together cause membrane damage bylipid peroxidation. Adrenergic signaling through PKA leads to inhibition of protein phosphatase 1. Together eIF2a kinase up-regulation and PP1down-regulation result in a rapid |20-fold increase in eIF2a(P), which inhibits initiator methionine introduction for peptide synthesis. CHOP transcriptionand caspase 3 activation are induced by eIF2a(P) through unknown mechanisms. Lipid peroxidation inhibits anti-apoptotic growth factor survivalsignaling. Dephosphorylated Bad sequesters Bcl-2, with consequent liberation of Bax, which induces mitochondrial release of cytochrome c and caspase 9to interact with APAF1 for activation of caspase 3 and apoptosis. Thus, the neurons die from radical and proteolytic injury to which they are unable torespond because of failed growth factor signaling and loss of translational competence.

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 3

3. Apoptotic mechanisms are engaged in vulnerable ‘charge’ recovers rapidly and approaches normal within 15neurons during early reperfusion, and these mecha- min of reperfusion [8,9].nisms include modifications of translation initiation,activation of proteolysis, and activation of endonu-cleases. 2.2. Reperfusion injury and selective vulnerability

4. These events not only exacerbate damage to criticalmolecules during reperfusion but also impair the Negovskii [10] proposed that the extent of tissue injurycompetence of cellular repair processes. observed following ischemia might largely reflect damage

5. Neuronal competence for antioxidant defense, transla- incurred during reperfusion. Morphologic studies providedtion initiation, inhibition of apoptosis, and repair of direct support for this hypothesis [11,12] and also iden-damaged cellular organelles is regulated at a fun- tified SVNs (hippocampal hilar and CA1 pyramidal neu-damental level by signal transduction mechanisms rons and cortical pyramidal neurons in layers 3 and 5).involving growth factors. Based on conventional staining, reperfusion damage in the

SVNs is observed in two phases [13]. Cytosolic mi-crovacuolation is noted within the first 15 min of reperfu-Because multiple independently lethal mechanismssion, although a substantial degree of normalization is(radical damage, loss of translation competence, proteolyticobserved by 1 h of reperfusion. Morphologic evidence ofactivation, and induction of apoptosis) are involved inprogressive damage to these neurons is then seen duringpost-ischemic neuronal death, single drug intervention willthe following 6 h, and by 48 to 72 h the neurons havebe ineffective. Thus we must undertake a complex ex-disintegrated.perimental effort that will involve both classical characteri-

zation of neuronal and functional loss as well as assays ofbiochemical markers reflecting the success or failure of

2.3. Post-ischemic brain hypoperfusioninterventions against the specific injury mechanisms inorder to develop and validate an effective multi-drug

Capillary hypoperfusion was first shown qualitativelytherapeutic protocol.[14], and subsequent quantitative studies demonstrated thatcortex capillary perfusion was only 30% normal by 90 minof reperfusion following 10 to 20 min of global ischemia[7]. This phenomenon occurred without a change in2. Historical observations of major phenomena inintracranial pressure and was originally taken to representbrain ischemia and reperfusion‘secondary ischemia,’ thought to lead to failure of high-energy metabolism and neuronal death during reperfusion.Four major observations have provided the foundationHowever, by the mid-1980s it was clear that calciumfor investigation of brain injury by ischemia and reperfu-antagonists [7], superoxide dismutase and deferoxaminesion [7]: (1) rapid loss of high-energy phosphate com-[15], or U74006F [16] (a lipid peroxidation chain ter-pounds during ischemia followed by their recovery withinminator [17]) inhibited post-ischemic brain hypoperfusion,the first 15 min of reperfusion; (2) morphological evidencebut had little effect on neurologic outcome. Moreover,that most structural damage occurs during reperfusion,tissue high-energy charge, which approaches zero within 4especially in selectively vulnerable zones; (3) progressivemin of complete ischemia [7], recovers rapidly duringbrain hypoperfusion during post-ischemic reperfusion; andreperfusion following ischemia, and this recovery is appar-(4) prolonged suppression of protein synthesis in selective-ently not adversely affected by the post-ischemic hypo-ly vulnerable neurons (SVNs).perfusion [18–20]. This evidence argues that post-ischemichypoperfusion is an epiphenomenon of more fundamental

2.1. High-energy phosphate compounds biochemical events and is not likely to be itself involved inneuronal death that occurs during reperfusion.

The brain’s ATP supply is dependent on continuousperfusion and approaches zero within about 4 min ofcomplete ischemia [7]. This energy depletion is associated 2.4. Inhibition of brain protein synthesis duringwith depolarization and loss of the normal 10,000/1 reperfusion

21partition of Ca between the extracellular fluid and thecytosol. Depolarization and the increase in intracellular Protein synthesis in vulnerable neurons is substantially

21 21Ca (Ca ) sets off a chain of events including lipolysis suppressed during post-ischemic reperfusion [18,21,22].i

during ischemia followed by free fatty acid metabolism Protein synthesis is a fundamental cellular activity, and a? 2and generation of superoxide radical ( O ) during reperfu- substantial part of our discussion below will focus on the2

sion [7]. During post-ischemic reperfusion, even after mechanisms involved in inhibition of protein synthesisischemic periods up to 1 h, the high-energy phosphate during reperfusion.

4 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

3. Classical mechanisms implicated in selective to salvage neurons following complete global ischemia.1vulnerability Furthermore, H (accumulated during ischemia) inhibits

the NMDA receptor [32], and there is little evidence forTwo major hypotheses emerged during the 1980s from persistently increased extracellular glutamate during re-

efforts to explain the phenomenon of selective vulnerabili- perfusion. These observations raise the question of whetherty. One is the excitotoxic neurotransmitter hypothesis ‘excitotoxicity’ at NMDA receptors is causally related todirected largely at events during ischemia, and the other is morphologic evidence of damage progression during re-the free radical hypothesis directed largely at events during perfusion; there is better evidence that AMPA receptorreperfusion. blockade is neuroprotective following complete global

ischemia [33]. In any case, glutamate excitotoxicity seemsonly to provide special mechanisms in SVNs for ischemia-

213.1. Excitotoxic amino acid neurotransmitters induced Ca overloading, which occurs anyway due tothe large transmembrane ionic gradient and ATP depletion-

The first of these suggests that SVN cell bodies and induced failure of the calcium pumps. Perhaps the mostdendrites receive projections that release large amounts of important thing about this hypothesis is that it representsamino acid neurotransmitter (i.e. glutamate) during is- an early insight that cell signaling mechanisms are in-chemia-induced depolarization [23]. There are two classes volved in neuronal death.of glutamate receptors, the ionotropic receptors, which arethe ligand-gated ion channels, and the metabotropic gluta- 3.2. Lipolysis, free radicals, and lipid peroxidationmate receptors (mGluR), which are coupled to cellulareffectors via GTP-binding proteins. On SVNs there are Free fatty acids (FFAs), and in particular free arach-two subtypes of glutamate ionotropic receptors that are idonic acid, are released during brain ischemia as adistinctively activated by either N-methyl-D-aspartate consequence of the activity of both phospholipase C(NMDA) or a-amino-3-hydroxy-5-methyl-4-isoxazole pro- (activated by depolarization) and phospholipase A (acti-2

21pionic acid (AMPA). NMDA receptor activation opens a vated by increased Ca ) [35,36], and elevated concen-i21 21Ca channel, allowing Ca influx, and AMPA receptor trations of these FFAs persist into the early recirculation

1 1activation opens a Na channel, allowing Na influx. The phase [34,37–42]. The rate of lipolysis during ischemia in1 1increased intracellular Na concentration (Na ) is thought the selectively vulnerable zones is significantly greateri

21to induce reversal of normal Ca extrusion through a 3 than in other areas of the brain [42]. Reperfusion-initiated1 21Na /Ca antiporter, thereby again resulting in increased metabolism of free arachidonic acid may be the major21 ? 2Ca , which is thought to exert cytotoxic effects, as we source of O . Cyclooxygenase catalyzes the addition ofi 2

will discuss below. The mGluRs are coupled to multiple two molecules of O to an unsaturated fatty acid, like2

second messenger systems, including those involved in arachidonic acid, and produces prostaglandin PGG, whichactivation of phosphoinositide hydrolysis, phospholipase D is rapidly peroxidized to PGH with concomitant release of

? 2activation, regulation of cAMP formation, and ion (calcium O [7].2

and potassium) channel modulation. The mGluR agonists The morphological progression of injury during reperfu-have two unusual effects, (1) reduction of transmission at sion [10–12] led to the hypothesis [43] that acceleratedexcitatory amino acid (glutamate) and inhibitory synapses structural damage during reperfusion is a consequence ofand of inhibitory post-synaptic potentials (probably via excessive generation of oxygen radicals followed by lipiddecreased GABA release) in the striatum and hippocam- peroxidation. The free radical hypothesis suggests that thepus, and (2) in the hippocampal CA region induction of SVNs are especially prone to radical-induced damage1

long-term synaptic potentiation, thought to be mediated by (specifically lipid peroxidation) during reperfusion becauseprotein kinase C-induced increases in NMDA receptor (1) they are deficient in glutathione peroxidase [44] andcurrents [24]. Intraocular injection of metabotropic re- (2) they are surrounded by iron-laden supporting cells thatceptor agonists protected against retinal damage induced release this iron during ischemia and reperfusion [45,46].by NMDA, but intra-hippocampal injections of mGluR The changes in the conformation of the fatty acidsagonists caused massive hippocampal damage [25,26]. resulting from lipid peroxidation would then alter the

Both glutamate release [23] and massive increases in permeability and fluidity of the membrane. In addition, and21Ca [27] (from |70 nM to|30 mM) occur during probably more importantly, membranes contain receptors,i

21complete global ischemia, and the Ca influx itself ion channels, and other proteins whose functions are likelystimulates glutamate release from presynaptic vesicles [7]. to be compromised by alterations in the lipid membrane.Re-uptake of glutamate is inhibited by arachidonic acid Lipid peroxidation is a set of radical-mediated chemical[28] or products of lipid peroxidation [29]. Although chain reactions whereby the double bonds in the polyun-magnesium, which inhibits synaptic transmitter release and saturated fatty acid (PUFA) side chains are rearrangedis a natural calcium antagonist, inhibits cell death in vitro [47,48]. In the presence of a transition metal (such as[30,31], NMDA receptor antagonists have generally failed ferrous iron), lipid peroxidation chain reactions can expand

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 5

geometrically [49]. The brain glia have abundant stores of ates oxygen radicals that result in cell death due tooxidized (ferric) iron [50], mostly in ferritin and transferrin subsequent lipid peroxidation, we have found that arach-

? 2[51]. Although O is not itself a potent oxidizer, it does idonate-induced cell death does not require radical mecha-2

promote reduction of ferric and release of ferrous iron nisms [77]. In neuronally-differentiated NB-104 cells, cellfrom ferritin [52], and iron delocalization into low-molecu- death caused by cumene hydroperoxide (a source oflar-weight species is seen in the brain during post-ischemic alkoxyl radical) is completely inhibited by either radicalreperfusion [45]. Peroxynitrite, formed by the reaction of scavengers or iron chelation, but cell death induced by? 2O with NO [produced by neuronal constitutive (nNOS) arachidonate is not inhibited by these strategies. The2

or inducible (iNOS) nitric oxide synthetase], is implicated radical-independent cell death induced by arachidonate is21as the lipid peroxidation-initiating radical species during associated with a prompt increase in cytosolic Ca

reperfusion [53,54]. Peroxynitrite-mediated nitrosylation of derived from both intracellular stores and the extracellulartyrosine residues is also seen in reperfused SVNs [55]. fluid; cumene hydroperoxide does not induce early release

21Membrane lipids are extensively peroxidized by iron- of intracellular Ca stores. These results show that,dependent radical reactions during reperfusion [46,56–60]. although radical damage can be lethal to this neuronal cellFormation of PBN (a radical spin trap) adducts peaks at 5 line, there is also a non-radical mechanism of arach-min reperfusion following 10 or 20 min of carotid occlu- idonate-induced toxicity associated with early release ofsion in rats, and total PBN adduct increases with increasing calcium from intracellular stores, an issue further consid-ischemic time [59]. Lipid peroxidation generates PUFA ered below.hydroperoxides and alcohols that are subsequently de-graded into several aldehydic products, including malon- 3.3. What is wrong with the excitotoxic neurotransmitterdialdehyde [61] and 4-hydroxynonenal [62]. Such lipid and free radical hypotheses?peroxidation products are seen by 15–30 min reperfusion[59,63], persist for up to 72 h [58], and are associated with We note that, because they are largely concerned withsubstantial loss of PUFAs, development of gaps in the two different phases of injury (i.e., ischemia vs. reperfu-plasmalemma, and failure of ion partitioning [12]. These sion), the glutamate and free radical hypotheses are notmarkers of progressive membrane damage are substantially mutually exclusive. There is evidence for the occurrence ofinhibited by pharmacologic iron chelators [64]. Three calcium entry mediated by both energy depletion anddifferent histochemical techniques have shown localization glutamate receptor activation during ischemia. Further-of lipid peroxidation to SVNs during reperfusion [65–67]; more, substantial evidence exists for membrane damagethe lipid peroxidation products appear to accumulate in the caused by both calcium activation of lipases and freeGolgi apparatus, an important site of membrane recycling radical generation during reperfusion. Unfortunately treat-[68,69]. ments directed only at these injury mechanisms do not

There is also evidence of continuing lipolysis during work.reperfusion [70]. Enzymatic lipolysis and lipid peroxida- Attempts at improving post-ischemic neurologic out-tion may act synergistically in membrane destruction come by blocking NMDA receptors have been frustratingduring reperfusion. For instance, erythrocytes damaged by [6,78], even though there is convincing evidence linkinglipid peroxidation are lysed more rapidly by phospholipase NMDA receptor excitotoxicity mechanisms to both mem-

21than are normal membranes [71,72]. Thus, fatty acids that brane damage by Ca -mediated mechanisms and radicalhave been peroxidized are better substrates for phospholip- induction [79]. Likewise, experimental therapeutic inter-ase. Moreover, some of the products of lipid peroxidation ventions that substantially inhibit lipid peroxidation duringstimulate the activity of phospholipase [73,74], and the reperfusion have had marginal effects on neurologicactivities of the lipid repair enzymes lysophosphatidylcho- outcome or morphometric evidence of cell death. Bio-line acyltransferase and fatty acyl CoA synthetase are chemical markers of damage by lipid peroxidation aresubstantially inhibited during reperfusion [75]. In addition, inhibited by deferoxamine [64]; however, deferoxaminelipid hydroperoxides interfere with the reacylation (salvage failed to substantially improve survival of SVNs [80].pathway) of neuronal phospholipids [76]. Similarly, treatment with deferoxamine combined with

Lipid peroxidation also appears to be involved in the superoxide dismutase [15] did not demonstrate majorgenesis of the post-ischemic hypoperfusion phenomenon, improvements in neurologic outcome, although a study ofwhich is inhibited by superoxide dismutase and deferox- hetastarch-complexed deferoxamine suggested a marginalamine [15] or U74006F [16], a lipid peroxidation chain improvement [81]. A report of improved neurologic out-terminator [17]. Thus, there is now evidence implicating come in post-arrest dogs treated with U74006F [82], amembrane damage by reperfusion-induced lipid peroxida- 21-aminosteroid that acts as a radical chain reactiontion in both histological damage to neurons and in the terminator, could not be confirmed [83]. Similarly, wegenesis of the post-ischemic hypoperfusion syndrome. were unable to demonstrate any improvement in neuro-

However, although this discussion suggests that during logic outcome with ethylmethylhydroxypyridone (EMHP)reperfusion oxidative metabolism of arachidonate gener- [84], a small lipophilic iron chelator.

6 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

The failure of therapeutic approaches directed at bloc- calpain is found primarily in CA1 and CA3 pyramidal cellsand in dentate gyrus granule cells, and the highest con-kade of excitotoxic neurotransmitters or free radical mech-centration of calpastatin is in ischemia-resistant pyramidalanisms indicates that these hypotheses do not yet adequate-cells of the CA3 region [100]. Calpastatin immunoreactivi-ly encompass the underlying mechanisms involved inty is present in both neurons and glia with diffuse stainingdeath of vulnerable neurons. We will suggest that otherof the cytosol and more pronounced staining of the cellimportant mechanisms involve terminal differentiation inmembrane [101].neurons, calpain-mediated proteolysis, fundamental altera-

The calpain system appears to regulate neurite out-tions in the systems that regulate protein synthesis, apo-growth, long-term potentiation, and synaptic remodeling inptotic mechanisms, and the effects of growth-factor signalthe brain [102,103]. These processes all involve alterationstransduction on many of these phenomena.of the plasmalemma and suggest that under physiologicconditions calpain activity is tightly regulated by local

21 21alterations of Ca concentration while the general [Ca ]4. Neurons and terminal differentiation i21remains normal. In contrast, the general increase in [Ca ]i

Regulation of the cell cycle may affect resistance to and that occurs during brain ischemia is likely to overwhelmrepair of membrane damage and be important in surviving endogenous regulatory systems and thus result inischemia and reperfusion. Non-replicating cells, such as pathologic calpain activity. Observations of proteolyticthose in the glomeruli, the myocardium, and the central degradation of calpain substrates were utilized to indirectlynervous system, are quite sensitive to damage by ischemia infer pathologic calpain activation in the brain in situationsand reperfusion. The linkage of DNA replication and including excitatory amino acid toxicity [104], ischemicmembrane synthesis follows from the requirement for both neuronal injury [105–110], Alzheimer’s disease [111], andfor cellular reproduction. In prokaryotes, experimental apoptosis [112].manipulations that specifically inhibit lipid synthesis block Both calpain isoforms share a common 30 kDa regula-DNA replication [85–87], and blocked DNA replication is tory subunit but have unique 80 kDa catalytic subunitsassociated with down-regulation of phospholipid synthesis with only 50% sequence homology [113,114]. Both the 80[88]. In human promyelocytes, sterol and phospholipid kDa and 30 kDa subunits of m-calpain and m-calpain (in

21synthesis are markedly decreased following induction of the presence of appropriate [Ca ]) undergo auto-myeloid differentiation [89], and there is a 90% decrease in proteolysis, which significantly reduces the calcium re-neuronal cholesterol synthesis following completion of quirement for subsequent proteolytic activity [97,115,116].differentiation in fetal rats [90]. Tumor cells, which have This autoproteolysis reduces the mass of the m-calpain 80their replication and anti-apoptotic signaling machinery kDa subunit first to 78 kDa and then to 76 kDa [117]. Inswitched on, are not easily killed by lipid peroxidation the presence of adequate calcium concentrations in vitro,[91], and the degree of differentiation is related to suscep- calpains will autoproteolytically degrade into inactive

21tibility to radical oxidant-induced death in many cell lines fragments. The [Ca ] required for autoproteolysis of m-[92]. Thus it is not surprising that there is a substantial calpain is about 10 mM but for m-calpain is about 500 mM,

21enhancement of radical-induced death in Neuroblastoma and for both m-calpain and m-calpain the [Ca ] required21B104 (NB104) cells after neuronal differentiation, and for autoproteolysis is slightly higher than the [Ca ]

differentiation-enhanced ultrastructural damage specifically required for subsequent proteolytic activity. Therefore,includes altered Golgi morphology similar to that observed autoproteolysis is universally accompanied by activation ofin vulnerable neurons of reperfused brains [93]. proteolytic activity [115,118,119]. Phospholipids, particu-

larly phosphatidylinositol (PI), lower the calcium require-ment for autoproteolysis of both m-calpain and m-calpain

5. Ischemia-induced calpain activation by five- to 10-fold [120]. Polyphosphatidylinositol 4,5-bisphosphate (PIP ) allows m-calpain to undergo auto-2

Calpains (EC 3.4.22.17) are a family of non-lysosomal proteolysis at calcium concentrations #1 mM [120].21neutral cysteine proteases [94,95] whose proteolytic activi- The differences in [Ca ] required for autoproteolytic

ty is absolutely dependent on calcium but is also regulated activation of the two isoforms predict autoproteolysis ofby phospholipids, by a specific endogenous inhibitor m-calpain but not m-calpain during brain ischemia, and the(calpastatin), and by a specific activator protein [96]. immunohistochemical localization of m-calpain predictsCalpains and calpastatin have been found in all vertebrate ischemia-induced activity of this protease may be mosttissues studied [97]. The two ubiquitous calpain isoforms important in the SVNs. In the section below on the effectsare m-calpain (calpain-I, CANP-I) and m-calpain (calpain- of ischemia and reperfusion on protein synthesis, we willII, CANP-II). Immunohistochemical studies have demon- see autoproteolytic activation of m-calpain during brainstrated that m-calpain is concentrated in neurons (soma and ischemia [121] and evidence that m-calpain degrades some

7dendrites) compared to glia, while m-calpain is more of the proteins involved in introduction of m G-cappedconcentrated in glia [98–100]. In the hippocampus, m- mRNAs for translation initiation. In the section below on

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 7

apoptosis, we will see evidence for early activation of ischemic preconditioning involves activation of Ras-me-caspase proteases by brain ischemia and reperfusion, and diated signaling.there is now evidence that m-calpain is involved in Although the patterns of post-ischemic protein synthesisactivation of caspase 7 by proteolytic processing of its suppression in the brain are well described, the mecha-proenzyme [122]. These findings show that the increased nisms that cause it were completely unknown until recent-

21[Ca ] associated with brain ischemia activates a calcium- ly. The integrity of DNA [134–136], transcription machin-i

dependent protease as well as phospholipases, and activa- ery [137–142], mRNA processing and transport [143–tion of calpain appears to be involved in both alterations of 145], and translational competence of purified ribosomestranslation initiation mechanisms and activation of apo- [146] are preserved in early reperfusion, and there isptotic pathways. adequate recovery of high-energy phosphates to support

peptide synthesis [147]; thus, these components are notresponsible for the inhibition of protein synthesis. Al-though there has not been a systematic study of the effects

6. Alterations in systems regulating protein synthesis of brain ischemia and reperfusion on intracellular levels ofall amino acids, the levels of those that have been

6.1. General considerations examined are not substantially reduced [148]. Additionalinvestigation is in order here because levels of aminoacyl-

Post-ischemic suppression of protein synthesis was first tRNAs do fall to |60–70% of control values during earlyreported in 1971 by Kleihues and Hossman [123], who reperfusion [127], and we will see below that unchargedobserved a 30% decrease in labeled amino acid incorpora- tRNAs can be involved in kinase activation directed attion after 4 h reperfusion following 30 min of ischemia. In inhibition of translation initiation by modification ofthe SVNs the post-ischemic suppression of brain protein eukaryotic initiation factors (eIFs).synthesis is prompt, severe, and prolonged. Cooper et al. Several lines of evidence are consistent with the hypoth-

14measured [ C]-phenylalanine incorporation by in vitro esis that protein synthesis is suppressed by inhibition oftranslation utilizing the post-mitochondrial supernatant translation initiation during early reperfusion. Ribosomalfrom rat brain homogenates [124]. Incorporation was sedimentation profiles obtained from reperfused brainsnormal in the homogenates derived from rats after 15 min show a preponderance of monomeric ribosomal subunitsof ischemia, but had fallen 80% after 15 min of reperfu- [18,123], suggesting a block in the formation of thesion. Nowak et al. found that in vitro amino acid incorpo- initiation complex [149]. In the study by Cooper et al. of inration by the post-mitochondrial fraction prepared from vitro translation by brain homogenates, the addition of agerbil brain homogenates was normal after 30 min of chain initiation inhibitor (polyinosinic acid) decreasedischemia without reperfusion [125]. However, after 10 min protein synthesis 63% in samples from control animals asof recirculation, in vitro protein synthesis was decreased expected [21]. However, polyinosinic acid had no effect on90% but recovered to 60% of baseline by 6 h of reperfu- in vitro translation with the postmitochondrial supernatantsion. These studies showed that as little as 3 min of obtained after 15 min of reperfusion, indicating thatischemia triggers suppression of protein synthesis during translation initiation was already maximally inhibited. Wereperfusion. The degree of suppression was dependent on extended this observation by showing that global brainthe duration of the ischemia for ischemic periods of less ischemia (10 min cardiac arrest) and 90 min reperfusionthan 5 min; longer ischemic times did not result in any reduce by 90% the rate of in vitro-initiated translation byadditional translation suppression but delayed translational brain homogenates [150].recovery in the surviving areas [22].

Translation competence in the post-ischemic brain is not 6.2. Formation of the translation initiation complexregionally homogeneous; the cortex, hippocampal CA1and hilus, and caudate show severe and prolonged suppres- The assembly of the initiation complex is an intricatesion of protein synthesis, whereas the hippocampal dentate process involving over 140 proteins, including at least ninegyrus and brainstem structures are less affected [126–131]. initiation factors in the eukaryotic cell [151]. Formation ofIn several of these studies the CA1 zone, which is most the initiation complex requires free 40S ribosomalsusceptible to neuronal death following ischemia [132], subunits; however, under normal physiologic conditions,never recovered protein synthesis, indicating that a pro- formation of inactive 80S ribosomes is favored. Binding oflonged deficit in post-ischemic protein synthesis correlates eIF6 to the 60S subunit and of eIF3 plus eIF4C to the 40Swith selective vulnerability. Furuta et al. showed that subunit act to keep the subunits disassociated. The eIF4improved neuronal survival following a substantial is- complex introduces mRNAs onto the small ribosomalchemic insult could be induced by brief periods of prior subunit. The eIF4 complex includesischemia, and that this tolerance was associated with earlyrecovery of protein synthesis in the hippocampal CA1

neurons [133]. We will see below that this phenomenon of 1. eIF4G, which provides docking sites for the other eIF4

8 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

components and for attaching the eIF4 complex to ribosome by an eIF4E-independent mechanism favored byeIF3 on the small ribosomal subunit, proteolytic fragments of eIF4G, and several viruses (in-

72. eIF4E, which binds m G-capped mRNAs, and cluding that for polio) utilize proteolysis of eIF4G and3. eIF4A, which is a helicase that unwinds the mRNA IRES-containing messages to favor translation of viral

near the 59 cap structure. proteins. Similar fragmentation of eIF4G occurs in stressedcells undergoing apoptosis [164], and this proteolysis of

Next, methionine attached to its special initiator tRNA eIF4G has the potential to affect selection of proapoptotic(Met-tRNA ) joins an eIF2-GTP complex to form the messages.i

‘ternary complex’ (Met-tRNA -eIF2-GTP) which then Our studies of brain ischemia and reperfusion have nowi

binds to the free 40S subunit. The 48S ribosomal complex identified two important changes in eIFs: (1) there is(including the 40S ribosomal subunit, the ternary complex, substantial proteolytic degradation of eIF4G mediated byand the mRNA-eIF4 complex) then ‘scans’ the mRNA in m-calpain during both ischemia [165] and reperfusionthe 59 to 39 direction to locate the AUG start codon. Once [150], and (2) there is a rapid |20-fold increase inthe appropriate match is made, eIF5 triggers the hydrolysis eIF2a(P) during reperfusion [150]. Autoproteolytic activa-of the eIF2-bound GTP and release of an eIF2-GDP tion of m-calpain occurs during ischemia in associationcomplex, and eIF4D then brings about a conformational with degradation of eIF4G that is blocked by calpainchange such that the 60S subunit joins the 48S subunit to inhibitors [165].complete the 80S initiation complex. In non-ischemic brain homogenates |1% of total eIF2a

The overall rate of translation initiation is regulated by is eIF2a(P); however, after a 10 min cardiac arrest and 90phosphorylation of serine-51 on the a-subunit of eIF2 min reperfusion (but not after ischemia alone), eIF2a(P) is[eIF2a(P)] [151]. The eIF2-GDP complex released during increased to |23% of total eIF2a [150,166]. This alterationinitiation must be recycled to eIF2-GTP; this is accom- occurs rapidly; at 10 min reperfusion eIF2a(P) is alreadyplished by exchange of the GDP for a new GTP. This increased |18-fold, and there is prominent eIF2a(P)transaction is catalyzed by the guanine nucleotide ex- immunostaining in the cytoplasm of neurons in both thechange enzyme eIF2B. Three key facts about this exchange hippocampus and cortex [166]. At 1 h reperfusion,reaction provide the basis for regulation of the overall rate eIF2a(P) remains only in SVNs [166] and is now promi-of translation by the amount of eIF2a(P): nent both in their cytoplasm on ribosomes and in their

nuclei in association with both the nucleolus and chromatin[167]. The nuclear translocation probably reflects the

1. GDP cannot be released from a-subunit-phos- nuclear localization motif (RRRIR) immediately adjacentphorylated eIF2 by eIF2B. to the ser-51 phosphorylation site on eIF2a, and a-subunit

2. a-Subunit-phosphorylated eIF2 has a 150-fold greater phosphorylated (but not unmodified) eIF2 binds DNA inaffinity for eIF2B than does unphosphorylated eIF2 electromobility shift assays [168] and may have important[152]. consequences for transcription. Immunohistochemical

3. In the brain, eIF2 is present in about a five-fold excess staining of eIF2a(P) and inhibition of in vivo protein35over eIF2B [153,154]. synthesis (by autoradiography of S-labeled amino acid

incorporation) co-map in the reperfused brain [169], andThus, a-subunit-phosphorylated eIF2 is a competitive insulin can induce dephosphorylation of eIF2a and full

inhibitor of eIF2B, and when .20% of eIF2 contains a recovery of protein synthesis in SVNs by 90 min reperfu-phosphorylated a-subunit, regeneration of the eIF2-GTP sion after a 10 min cardiac arrest [169]. These resultscomplex and delivery of the first amino acid for all new provide a mechanistic basis for the inhibition of proteinprotein synthesis is severely inhibited. synthesis in SVNs during reperfusion, and together with

The issue of message selection is complex and not other evidence of a role for eIF2a(P) in the mechanisms ofcompletely understood. Normally, the cell appears to apoptosis (discussed below) suggest that the inhibition ofregulate which mRNAs bind to the ribosome by modu- protein synthesis in SVNs during reperfusion may be a

7lating phosphorylation of the m G-cap-binding protein fundamental phenomenon in the causal pathway leading toeIF4E [155]. Availability of eIF4E is limited in cells [156], the death of vulnerable neurons.and the selection of mRNAs for translation (such as thosefor protooncogenes) can be modulated by phosphorylation 6.3. Implications of eIF modifications induced byof eIF4E [157]. eIF4E is phosphorylated on serine-53 in ischemia and reperfusion for message selectionresponse to several growth factors including insulin [158],PDGF [159], NGF [160] and epidermal growth factor Both the proteolytic degradation of eIF4G and the[161]. Multiple sites on eIF4G are phosphorylated, and this phosphorylation of eIF2a are likely to have importantis thought to induce increased binding of eIF4E [162] and implications for message selection for residual proteinenhanced protein synthesis [163]. Uncapped mRNAs con- synthesis in SVNs. The response of translation initiation totaining internal ribosome entry sites (IRES) can enter the heat shock and to reperfusion in SVNs shares some

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 9

common elements, including phosphorylation of eIF2a overall protein synthesis in SVNs during reperfusion and[170,171]. However, the response to heat shock is not suggestions that administration of cyclohexamide (ancompletely analogous to that seen after ischemia; although inhibitor of peptide elongation) reduces what is thought todephosphorylation of eIF4E is seen in heat shock [172– be apoptotic degeneration of SVNs [184,185].174], we did not find dephosphorylation of eIF4E inducedby brain ischemia and reperfusion [150]. Joshi-Barve et al. 6.4. eIF2a kinases and phosphataseshave shown that antisense-induced depletion of eIF4E andeIF4G substantially inhibits protein synthesis, but about 20 There are four mammalian eIF2a kinases: (1) GCN2proteins, including HSP-70, continue to be synthesized in [186], (2) the hemin-regulated inhibitor in reticulocytesthis situation [175]. In the situation of brain ischemia and (HRI) [187], (3) ‘RNA-activated’ protein kinase (PKR)reperfusion, proteolytic loss of eIF4 components is there- [187], and (4) PERK (PKR-like ER kinase) [188]. We dofore inadequate to explain the blocked translation of HSP- not yet know which one is responsible for phosphorylation70 [176,177] in SVNs, which in fact is almost certainly a of eIF2a during brain reperfusion.consequence of the large increase in eIF2a(P). GCN2 was first identified in yeast, where it is activated

The translation of all messages is not uniformly in- by amino acid starvation [186]. Recently, GCN2 analogueshibited by increased concentrations of eIF2a(P). An have been identified in the CNS of Drosophila [189] andimportant example of this is the case of the message for mice [190]. Three mammalian GCN2 isoforms are gener-GCN4 in yeast [178]. GCN2 is the yeast eIF2a kinase and ated by alternative splicing of the sequence for the N-is activated by uncharged tRNAs caused by amino acid terminal region [190], and all three isoforms are strongly(specifically histidine) deprivation. GCN4 is a transcription expressed in the mouse brain [190]. GCN2 appears to befactor for a family of enzymes for de novo amino acid constituitively associated with and down-regulated bysynthesis. Activation of GCN2 is followed by increased HSP90 [191], and geldanamycin, which has been shown toeIF2a(P) and enhanced translation of message for GCN4 protect neurons from glutamate-induced oxidative damage[178]. The message for GCN4 is polycistronic with a long and apoptosis [192], stabilizes the association of GCN259 leader containing three open reading frames (ORFs) and HSP90 [191]. There are at least three potentialfollowed by the actual code for GCN4 in the 4th and most ischemia and reperfusion-induced activation mechanisms39 ORF. In the absence of eIF2a(P), the ORF nearest the for GCN2. Although there are no published observations59 leader is translated and GCN4 is not synthesized. of the effects of brain ischemia and reperfusion on levelsHowever, in the situation of the marked reduction in the of aminoacylated histidine tRNA, histidine loss occurs inavailability of eIF2-GTP complexes imposed by eIF2a(P), association with brain ischemia [193,194], and histidinethe ribosome is less likely to be able to initiate translation administration offers some neuroprotection [195]. Second,at the upstream ORFs and continues scanning [178]. The a cytoplasmic unfolded protein response, consistent withprobability of a ternary complex binding to the small HSP transcription, could induce dissociation of HSP90ribosomal subunit is enhanced over longer scanning times, from GCN2 and activation of the enzyme. Finally, Rolfesand thus GCN4 gets translated because initiation does not and Hinnebusch [196] found that GCN2 is also activatedoccur on the ORFs nearer the 59 end of the message [178]. by low adenine levels, known to occur in response to brain

These considerations suggest that translation of proteins ischemia and reperfusion [197]; however, their work didencoded by 39-distal ORFs on mRNAs with IRES and long not rule out the simple possibility that, in their model,59 leaders containing other ORFs might actually be favored purine deficiency reduced tRNA charging as a conse-in the circumstance of degradation of eIF4G and increased quence of ATP deficiency.eIF2a(P). Several examples of such mRNAs are relevant HRI activity is stimulated by heavy metals (e.g., iron)to the problems associated with brain ischemia and re- [198], oxidized glutathione, and polyunsaturated fatty acidsperfusion. The mRNA for eIF4G itself has a long 59 leader and lipoperoxides [199]. These activation mechanismsand contains multiple ORFs [179] and an IRES [180], and suggests a potential causal link between radical-mediatedtransfer of this 59 leader onto reporter genes can increase iron delocalization, lipid peroxidation, and the reperfusion-their translation over 100-fold [179]. Thus, the levels of dependent suppression of protein synthesis. However, PaleIF4G should be able to recover during reperfusion. Other et al. could not find HRI in rabbit brain sections [200], andimportant mRNAs that have recently been found to also we have also found little HRI in brain homogenates byhave these characteristics include those for apoptosis-ac- Western blotting. Furthermore, HRI-knockout mice (kindlytivating factor-1 (APAF-1) [181], vascular endothelial provided by J.J. Chen, Harvard-MIT, Cambridge, MA,growth factor [182], and fibroblast growth factor-2 [183]. USA) show no reduction in brain eIF2a phosphorylationApoptosis, in particular, is a crucial area in which these during reperfusion (DeGracia et al., unpublished data).issues are likely to be important. The above considerations PKR is widely distributed in various tissues, includingsuggest that careful examination of the products of residual the brain [201]. PKR was first identified as a double-translation in SVNs during reperfusion may resolve the stranded RNA-activated eIF2a kinase [202]. Its majorapparent conflict between the biomolecular inhibition of function is thought to be to oppose viral infection [203],

10 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

although recent observations indicate a broader role for antisense strategies in conjunction with rat cardiac arrestPKR in cellular events [203,204]. PKR is increased in and resuscitation.differentiated cells [204], and a number of endogenous The greatly enhanced serine-51 phosphorylation oninhibitors and activators of PKR have been identified eIF2a during brain reperfusion could also reflect inhibition[205]. Over expression of either inactive mutants of PKR of the phosphatase activity against eIF2a(P). Althoughor endogenous inhibitors of the enzyme result in tumori- Kimball et al. found decreased eIF2a(P) phosphatasegenesis, suggesting that PKR may be a tumor suppressor activity in the liver after amino acid deprivation [221], we[205]. Furthermore, activation of PKR has been correlated did not observe diminished phosphatase activity againstwith changes in gene expression; it is particularly interest- eIF2a(P) in reperfused brain homogenates [211]. How-ing that activated PKR appears to be required for induced ever, the issue of phosphatase activity is certain to betranscription of the gene for the FAS receptor involved in complicated by subcellular targeting issues.apoptosis [206,207]. PKR is found tightly associated with Ser-Thr protein phosphatases are grouped into fourribosomes [187] and on the rough ER [208] with a small major classes based on their catalytic subunits and respon-fraction present in the nucleolus [209]. PKR transcription siveness to various inhibitors [222]. PP1 and PP2A de-is induced by interferon, and PKR activity is antagonized phosphorylate eIF2a(P) in vitro, but neither PP2B norby Ras-activated cell signaling processes [210], such as PP2C do so [223]. This is also the case in cells [224]those known to be under the control of the insulin receptor. where addition of okadaic acid [222] (an inhibitor of PP1

2 / 2However, we have shown that PKR mice show no and PP2A) results in shutdown of protein synthesis.reduction in the accumulation of eIF2a(P) induced by Furthermore, PP1, but not PP2A, is specifically inhibitedbrain ischemia and reperfusion [211]. by two peptides known as inhibitors 1 and 2 (I-1 and I-2)

PERK is an eIF2a kinase that responds to ER stress [222], and in the reticulocyte lysate addition of either I-1[188]. Many nascent peptides during their synthesis enter or I-2 leads to increased eIF2a(P) and inhibition ofthe ER to undergo processing. ER-resident chaperones translation [225]. Thus, PP1 is the dominant phosphatase(such as GRP78) and protein isomerases facilitate proper for eIF2a(P) [226]. There are four PP1 isoforms (a, g1,folding of the new peptides, and ER glycosylases carry out g2, d), with PP1 a, g1, and d present in the brain [227],post-translational processing on many new proteins [212]. and PP1 a in particular present in the neuronal perikaryalSeveral of these ER protein processing reactions require cytoplasm [228,229].

21physiologic ER Ca concentration (similar to that in PP1 activity is extensively regulated by signal transduc-21extracellular fluid), and depletion of Ca from the ER tion and molecular targeting mechanisms [230]. PP1

leads to an unfolded protein response (UPR) that in higher enzyme complexes consist of a 35 kDa catalytic unit (noweukaryotes includes a rapid suppression of protein syn- itself called PP1 [230] but previously designated PP1c)thesis [213–217] mediated by increased phosphorylation of combined with a wide variety of targeting and regulatoryeIF2a [218–220] that does not require PKR [188]. In the subunits. A bimetal center (Fe /Zn or Fe/Mn) is present inwell characterized yeast ER UPR [212], ER lumenal the PP1 active site and appears crucial for both bindingunfolded proteins are detected by IRE-1, followed by and catalytic removal of the target phosphate [230]. Thereactivation of its cytosolic RNAse. The IRE-1 RNAse is compelling evidence that PP1 functions only whenprocessing of Hac1 mRNA allows translation of this associated with one of |30 different targeting subunitstranscription factor for the ER chaperones and isomerases. (including the ribosome L5 protein), and known targetingPERK is an evolutionary recombinant protein with homol- subunits indicate PP1 regulation of glycogen metabolism,ogy to the IRE-1 unfolded protein detector in its N- protein synthesis, and cell cycle machinery [230]. The PP1terminal ER-resident domain and homology to GCN2 in its targeting issue can be crucial in preventing accumulationcytosolic eIF2a kinase domain [212]. PERK activation of eIF2a(P); although herpes virus activates PKR, ainvolves autophosphorylation, and the active form displays virally-expressed PP1 targeting unit with homology toretarded electrophoretic mobility [188]. PERK is present GADD34 causes an |10,000-fold increase in PP1 activityand activated in response to thapsigargin (a selective against eIF2a(P) and thus prevents PKR-induced shut-off

21inhibitor of the ER Ca pump [188]) or arachidonate in of translation initiation [231]. PP1 can also inactivate PKRneuronally-differentiated NB104 cells (O’Neil et al., un- [232] and by inference could be involved in down-regula-published data, PERK antibody kindly provided by David tion of the activity of other eIF2a kinases.Ron), and arachidonate induces both release of intracellular In general, signal transduction mechanisms do not

21Ca stores and phosphorylation of eIF2a in these cells induce post-translational modification of PP1 itself but[77]. Unfortunately, in our hands Westerns of brain rather regulate phosphorylation of PP1 targeting subunitshomogenates have substantially more ‘noise’ around and/or inhibitors [222,230,233]. In this regard PP1 andPERK than is present with homogenates from NB104 PP2A often appear to be counter-regulatory in signalingcells; this has obstructed identification of any PERK mechanisms in which growth factors and adrenergicmobility shift induced during brain reperfusion. We are agonists exert opposite effects. For example, in differen-therefore currently evaluating the role of PERK utilizing tiated myocytes insulin induces rapid doubling of PP1

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 11

activity against glycogen synthase through a site-specific different absolute concentrations of p67, and cells withphosphorylation on the appropriate targeting subunit, high p67 content exhibit enhanced resistance to viral-whereas PP2A dephosphorylates this targeting subunit site induced eIF2a phosphorylation [248]. Viral-induced phos-and thereby inactivates PP1 dephosphorylation of glycogen phorylation of eIF2a and shut-off of host-cell proteinsynthase [234]. PP2A itself is activated through the cAMP- synthesis is associated with rapid activation of a 60 kDadependent protein kinase A (PKA) but deactivated by an enzyme that deglycosylates p67 [247], which makes p67insulin receptor-induced tyrosine phosphorylation [235]. unable to prevent phosphorylation of eIF2a but does notRecently, two peptide inhibitors of PP2A have been lead to its degradation [245]. These findings suggested thatidentified and shown to enhance the activity of PP1 against p67 deglycosylation during ischemia and reperfusion and/a number of substrates in a manner dependent on a or the level of expression of p67 by various groups of

21physiologic concentration of Mn , and these investigators neurons could affect their susceptibility to eIF2a phos-have noted the potential of this system to account for phorylation. However, we have recently found that neitherseveral examples of PP1 and PP2A counter-regulation the glycosylation state of p67 nor its brain immuno-[236]. localization are affected by ischemia and reperfusion

Of the several known PP1 inhibitors [237], one [249]. Although altered p67 glycosylation does not appear(DARPP-32) is found primarily in basal ganglia rather to be in the causal pathway leading to phosphorylation ofthan in vulnerable hippocampal or cortical neurons, nearly eIF2a during post-ischemic reperfusion, the persistence ofnothing is known about the intriguing ribosome inhibitor glycosylated p67 may be important in limiting the extentof PP1 (RIPP-1), and NIPP-1 is in the nucleus and of eIF2a phosphorylation (which in our hands neverunlikely to be involved in any regulation of cytoplasmic exceeds 23% of total eIF2a) and supporting the potentialeIF2a(P). As we noted above, I-2 can inhibit PP1 activity, for recovery of translation competence in vulnerablebut emerging evidence suggests that I-2 may be more neurons.important as a folding chaperone than as an inhibitor ofPP1 [230,237]. Thus, here we focus on I-1, which is an 6.6. The importance of recovery of protein synthesis18.7 kDa cytosolic peptide that, when phosphorylated on during early reperfusionthreonine 35 by PKA, competitively inhibits PP1 with a Ki

of 1.6 nM [237,238]. The average concentration of I-1 (1.6 Late in reperfusion, other fundamental mechanisms maymM in skeletal muscle) is about twice that of all forms of serve to perpetuate the suppression of protein synthesis. ByPP1 combined. Epinephrine, isoproterenol, and dopamine 8 h of reperfusion following a 15 min cardiac arrest ininduce PKA activation of I-1; this effect is opposed by dogs, ionic partitioning is lost, associated with a concomi-insulin [234,239], which along with other growth factors tant 30% decrease of lipid double bond content andinduces increased PP1 activity [240]. I-1 is found mostly ultrastructural evidence of large breaches in the plas-in the pyramidal neurons in the hippocampus and layers malemma of SVNs [12]. The loss of ionic gradients of

1 1 21 21II–III and V–VI in rat cerebral cortex [241,242], and I-1 is Na , K , Ca and Mg are incompatible with transla-activated in hippocampal neurons by glutamate-induced tional competence [250–252] and cell viability. Accord-long-term potentiation [237]. Taken together these findings ingly, effective therapy will probably have to allowsuggest investigation for threonine 35 phosphorylation on recovery of protein synthesis before 4–8 h reperfusion inI-1 during brain reperfusion and raise the possibility that order to enable the vulnerable neurons to respond in waysinsulin’s post-ischemic neuroprotective effects (that are that will prevent the later deterioration.independent of any hypoglycemia [243]) and its ability to As one would expect, there is evidence that an irrevers-induce dephosphorylation of eIF2a(P) and restoration of ible suppression of protein synthesis represents a lethaltranslation competence could involve dephosphorylation of cellular lesion, and inability to express proteins assures cellI-1. death. Furuta et al. have demonstrated that recovery of

protein synthesis is probably a prerequisite to neuronal6.5. Regulation of the availability of eIF2 to eIF2a salvage [133], and Hossmann’s group has very recentlykinases demonstrated in a focal ischemia model that early failure

of protein synthesis (at 1 h ischemia) occurs in a penumbr-A final mechanism that might be involved in eIF2a al zone before ATP depletion and precisely demarcates the

phosphorylation during post-ischemic brain reperfusion is ultimate extent of the expanding lesion [253]. Someregulation of the availability of eIF2 as substrate for eIF2a investigators have reported a reduction in delayed neuronalkinases. Gupta and collaborators [244–247] have identified death with the administration of known inhibitors ofa peptide with M 67 kDa (p67) that, when glycosylated, protein synthesis such as cylcohexamide [184,185]; how-r

binds to eIF2 and protects eIF2a from phosphorylation, ever, it is not possible to conclude from these experimentsand they have shown that preincubation of brain homoge- that the inhibition of protein synthesis that develops as anates with antibody against p67 enhances subsequent in consequence of ischemia and reperfusion is directedvitro phosphorylation of eIF2a. Different cell lines contain toward neuron sparing. Indeed, these investigators did not

12 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

examine the effect of their intervention on protein syn- into their specialized positions, the excess cells are elimi-thesis by either autoradiography of tissue sections or nated by apoptosis. These genetically programmed pro-translation by brain homogenates. It is likely that identifi- cesses during development have provided a reproduciblecation and molecular elimination of the kinase responsible phenomena, and developmental apoptosis has been exten-for eIF2a phosphorylation during reperfusion will allow sively studied in Caenorhabitis elegans. Embryonic de-these issues to be settled by evaluation of neuronal survival velopment of this nematode produces exactly 1090 cells,when the reperfusion-associated inhibition of protein syn- of which 131 undergo apoptosis [258]. Studies of apop-thesis is prevented. tosis in this model reveal five groups of genes involved

with this process [255]: those triggering cell death, thoseinvolved in the cell death process itself (including ced-3

7. Apoptosis and ced-4, that is, C. elegans death gene), those required] ] ]

for engulfment of the dying cell, those implicated in final7.1. General considerations disposal of the cellular corpse, and a single gene (ced-9 )

involved in suppression of cell death. Mammalian genesApoptosis is a process of self-destructive cell death that have been identified with sequence homology to three of

involves activation of mechanisms encoded in the genomes those in the C. elegans system [255]; there are genes withof all higher eukaryotes [254]. Appreciation of the impor- homology to ced-3 [ICE-protease (interleukin 1b

]tance of apoptosis in mammalian systems has only de- converting enzyme and the more general family of caspase

] ]veloped during the last 5–7 years [255], and some of the proteases)], ced-4 (apoptosis-activating factor 1, APAF-1),details of the process remain poorly understood. Neverthe- and ced-9 (the Bcl-2 family).less, it has become clear that the process of apoptosis isintimately involved in several acute disease states (includ- 7.3. Receptor-mediated caspase activationing injury mechanisms involved in ischemia and reperfu-sion of organs), and mutational events leading to inhibition In higher eukaryotes several genes with homology toof apoptosis are involved in the development of many ced-3 have been identified. These include genes for thecancers. Furthermore, some prototypes of therapeutic FAS (also called Apo-1), TNFR (tumor necrosis factorapproaches to these processes are now emerging. Thus receptor), and TRAIL-R (tumor necrosis factor-relatedhere we will examine the signaling pathways and the apoptosis-inducing ligand receptor), plasma membranespecific execution mechanisms of apoptosis. receptors that activate intracellular signaling pathways

In early work, DNA fragmentation into size ‘ladders’ leading to apoptosis by activation of caspase proteasesobserved after electrophoresis was thought to be the [259,260]. These receptors include a cytoplasmic region ofhallmark of apoptosis. These ‘ladders’ were generated |70 amino acids, called the ‘death domain,’ that is abecause DNAse activity produced random double strand protein–protein interaction zone shared by many of thecuts in the internucleosomal regions. Increased intracellu- proteins in this system [255]. Ligand binding to these

21lar Ca appears to be involved in activation of this receptors leads to recruitment of adapter-linkers (FADD,DNAse(s) [256]. Subsequently it was recognized that there CRADD, TRADD, etc.) that then recruit upstream caspaseswere additional morphologic alterations characteristic of to the activation complex by association of death domainsapoptosis; these include chromatin condensation, extensive [261]. Caspase 8 (Mach1, FLICE) is an upstream caspasealterations in the microtubular array, fragmentation of the that is recruited to the receptor–adapter complex and thencell into ‘apoptotic bodies,’ and detachment of the cell cleaves other pro-caspases into the active proteases [262].from neighbors or culture substrate [254,255]. The altera- An important function of the apoptotic system is totions in a specific instance of apoptosis are somewhat provide a viral defense in which infected cells are inducedvariable, and it has now become clear that the cytoplasmic to die, and several viruses encode death-domain-containingalterations of apoptosis (and indeed cell death) can occur proteins that inhibit caspase-mediated apoptosis. Thiswithout the appearance of DNA fragmentation [257]. This inhibition is achieved by either binding caspase 8 (cowpoxvariability in the phenomenology of apoptosis raised protein CrmA [262,263] and herpes virus protein E8 [264])problems with respect to assays that allow its confident and blocking both its ability to associate with the receptor–recognition. adapter complex and to activate downstream caspases

[262] or by binding linker-adapters (molluscum con-7.2. Insights from apoptosis during embryonic tagiosum virus MC159 protein [264]) and inhibiting theirdevelopment of C. elegans function.

The relevance of this membrane receptor signalingApoptosis is an important process in embryonic de- system to reperfusion has recently been shown. FAS

velopment. Many more cells are generated than are finally protein is increased |130-fold in post-ischemic myocytesneeded, particularly in highly differentiated organs such as at 2 h reperfusion following myocardial ischemia [265],the kidney, heart, and brain [255]. As these cells migrate and there is now compelling evidence for the involvement

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 13

of apoptosis in the post-ischemic death of cardiac an amino-terminal-truncated IkBa lacking Ser-32 [273].myocytes [266]. The situation with regard to apoptosis This truncated IkBa cannot be phosphorylated on theinduction by ligand activation of FAS, TNFR, or TRAIL-R missing Ser-32, is not ubiquinated and degraded, andduring brain reperfusion is less clear. Although there is statically binds and inhibits NF-kB, which is then unablelittle FAS mRNA in normal brains and Matsuyama et al. to provide prosurvival transcriptional support. Althoughfound FAS mRNA induced by 6 h reperfusion following we found no studies of NF-kB activity or localization30 min ischemia in mice [267], this group’s mapping by in during the early minutes of brain ischemia and reperfusion,situ hybridization did not observe FAS expression by it has been shown that IkB is degraded and nuclear NF-kBvulnerable neurons during reperfusion. However, Martin- activity is increased within minutes of cardiac ischemiaVillalba et al. [268] have elegantly demonstrated co-locali- [281], and neuronal nuclear localization of NF-kB haszation of N-terminal phosphorylation of the transcription been observed at 24 and 72 h brain reperfusion [282,283].factor JUN (which can be induced by signaling from these NF-kB activity is lost in the ischemic core in strokereceptors [260]) and production of FAS ligand in neurons models, and inhibition of NF-kB activation considerablyadjacent to the ischemic core and provided evidence that enhances the sensitivity of neuronal cell lines to apoptosisFAS ligand can kill neuronal cell lines, an effect that can induced by TNFa [284]. Furthermore, although knockoutbe overcome by expression of a dominant negative mutant of the p50 component of NF-kB is not lethal, these miceof FADD [269]. Brain localization of TRAIL-R by im- are deficient in transcription factor binding to the NF-kBmunohistochemistry or in situ hybridization has not been consensus sequence and display substantially enhanced inreported, but brain injection of TRAIL is not neurotoxic, vivo injury of SVNs by excitotoxic mechanisms [285].although it can eradicate malignant glioma [270]. The The presence and role of death-inducing plasmalemmalpicture with respect to TNFR is similarly complicated; ligand receptors in brain ischemia and reperfusion requiresChopp’s group showed appearance of TNFR as quickly as further clarification, and the possibility remains that a1 h after MCA occlusion in mice, but the protein was receptor-activated caspase apoptotic response is not en-localized to microvessels [271]. Moreover, TNFR knock- gaged as a primary mechanism of death in the vulnerableout mice showed exacerbated brain damage in response to neurons. There are, however, intracellular mechanisms thatfocal ischemia [272], possibly because TNFR activation can engage the caspases independent of the receptor-also leads to activation of the transcription factor NF-kB activated mechanisms.[206], which can counter-regulate apoptotic mechanisms.

NF-kB is a transcription factor comprised of 50 and 65 7.4. Intracellular mechanisms of apoptosiskDa subunits and is normally associated with its inhibitorIkB, which binds and sequesters NF-kB in the cytoplasm 7.4.1. Non-receptor-mediated caspase activation[273]. Ligand-induced association of TNFR with tumor The caspase system is more complicated than just thenecrosis factor receptor-associated factor 2 (TRAF2) [274] well characterized FAS, FADD/CRADD, caspase 8 mech-induces activation of the IkB kinase complex, which anism reviewed above. Currently about 14 caspases havephosphorylates IkBa on Ser-32 and Ser-36 [273]. Phos- been identified and can be divided into ‘initiators’ andphorylation of IkBa on these sites is followed by its ‘effectors.’ The interactions with caspase-recruiting adap-ubiquination on Lys-21 and -22 and its degradation by the ters by long pro-domains in initiator caspases 8 and 9proteosome. This liberation of NF-kB from IkB allows activate the proteolytic function of these caspases, whichnuclear translocation of NF-kB, which acts as a transcrip- in turn activate other effector caspases by proteolysistion factor to induce expression of genes promoting cell [286]. This type of associative activation mechanism cansurvival, such as ‘cellular inhibitors of apoptosis’ 1 and 2 be set off inside the cell without involvement of plas-(c-IAP1 and c-IAP2) [275,276], A20 [277] (an anti-apo- malemmal receptors. For example, the prototypic caspaseptotic zinc-finger protein whose mechanism of action CED-3 can be activated by direct association with CED-4remains unknown [278]), and manganese-superoxide dis- [287], which undergoes nuclear localization and inducesmutase [279]. We note that because pro-survival NF-kB cell death that requires function of CED-4’s nucleotide-signaling is induced from TNFR and requires transcription binding P-loop [288]. Co-expression of CED-9 with CED-and translation, the use of translation inhibitors has been 4 blocks the nuclear localization and lethality of CED-4required in many studies of TNFR-induced apoptosis. The [288] by a direct interaction between these two proteinsc-IAPs appear to block both activation of caspase 8 at the [289].ligand receptor complex and the activity of proteolytically- In mammalian cells the homologue of CED-4 (apop-processed caspases [276], and vector-directed overexpres- tosis-activating factor 1, APAF-1 [290]) and its associativesion of the X-linked IAP blocks both caspase 3 activation initiator caspase (caspase 9) are normally in separateand death of hippocampal SVNs during brain reperfusion compartments; APAF-1 is cytosolic, while in SVNs both

2 / 2[280]. NF-kB p65 embryos die from massive apoptosis cytochrome c and caspase 9 reside in the mitochondrial[273]. Furthermore, during apoptosis caspase activity intermembranous space [291]. In the presence of dATPappears to lead to inactivation of NF-kB by generation of and cytochrome c, APAF-1 undergoes self-oligomerization

14 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

and activates caspase 9, which then activates caspase 3 and localization of specific caspases during post-ischemic[286]. Cytochrome c and caspase 9 are coordinately brain reperfusion.released in response to mitochondria undergoing mem-brane permeability transition or interaction with the pro- 7.4.2. Bcl-2, Bax, and Badapoptotic Bcl-2 homologue Bax (discussed below) [291]. In the C. elegans system, the protein encoded by ced-9SVN mitochondria release both cytochrome c [292,293] inhibits apoptosis. The human genes encoding CED-9-and caspase 9 [291] within 1–2 h following ischemia but related proteins include Bcl-2, Bax, Bad, Bcl-x, mcl-2, Bak,do not appear to undergo membrane permeability transition and Bak-2 [255]. In terms of the ‘viruses vs. eukaryotes[12,291,294]. Proteolytically-activated caspase 3 can be wars,’ it is interesting that several mammalian viruses,seen as early as 1 h reperfusion [295]. After release of including the Epstein–Barr virus (mononucleosis andcaspase 9 from SVN mitochondria, it is substantially Burkitt’s lymphoma), carry genes encoding Bcl-2 homo-translocated into SVN nuclei [291], consistent with other logues that inhibit apoptosis [315]. There is no evidenceobservations of an ‘apoptosome’ complex containing that Bcl-2 directly inhibits caspases, but it does appear to

21APAF-1, cytochrome c, and caspase 9 undergoing nuclear inhibit release of Ca from the endoplasmic reticulumlocalization during apoptosis [296]. (ER) [316], an event that we have already seen can induce

Although caspases are constituitively expressed as phosphorylation of eIF2a and that we will see can beproenzymes [262], ischemia and reperfusion also induce crucial to the activation of intracellular apoptosis mecha-transcription for caspase 3 in vulnerable neurons [297]. In nisms. The Bcl-2 gene encodes two proteins generated byvarious model systems caspase 3 is activated before alternative splicing products at the C-terminus, whichseveral other caspases [298]; indeed, caspase 3 is the affects membrane localization. Bcl-2a is localized to thepredominant enzyme involved in very rapid cleavage of outer nuclear membrane, the ER, and the outer mito-the DNA repair enzymes poly(ADP ribose) polymerase chondrial membrane; Bcl-2b is a cytosolic protein [255].(PARP) [299,300] and DNA-dependent protein kinase Both the Bad and Bax gene products are promoters of[301]. PARP recognizes strand nicks in DNA and marks apoptosis. Release of Bax from binding by Bcl-2 [317]them for repair by generating poly(ADP ribose) chains on allows Bax to oligomerize and form channels in mem-adjacent nuclear proteins [302], up-regulates the tumor branes [318], including the outer mitochondrial membranesuppressor p53 [303], is required for cellular recovery after where it can cause the release of cytochrome c [318] andradical-mediated DNA damage [304], and its activity is caspase 9 [291]. Homozygous deletion of Bax inducesrequired for insertion of retroviral-derived DNA into the substantial attenuation of post-ischemic neuronal deathgenome [305]. Caspase 3 also cleaves actin to produce a [319]. The tumor suppressor transcription factor p53 ischaracteristic 15 kDa peptide [306]. Caspase 3 is clearly activated in response to poly(ADP)-ribosylation of nu-involved in developmental apoptosis and is required for cleosomal proteins and both up-regulates Bax and down-normal brain development in mice [307]. Caspase 3 regulates Bcl-2 transcription [255]. These observationstranscripts are present in neurons of developing rat brains suggest that the ratio of Bcl-2 to Bax is important inbut are greatly down-regulated in the adult brain [308], regulating apoptosis.although some caspase 3 is constituitively expressed in the Bad appears to exert its pro-apoptotic effects by bindingadult brain [309]. There is evidence for active caspase 3 in and sequestering Bcl-2 [320]. The activity of Bad isSVNs within a few hours following cerebral ischemia regulated by phosphorylation; the dephosphorylated form[295,309], and the relatively specific caspase 3 inhibitor binds Bcl-2. Bad is phosphorylated on serines 112 and 136N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluromethylketone by Akt (PKB), a central element in survival signaling, thatdecreases caspase cleavage products, reduces tissue dam- is activated through the growth-factor-mediated PI-3 ki-age, and improves functional outcome in a stroke model in nase pathway [321]. Phosphorylated Bad is sequestered inrats and mice [310]. the cytoplasm by 14-3-3 proteins [296]. A major effect of

The details of non-receptor-mediated caspase mecha- maintaining Bad in its phosphorylated state appears to benisms involved in an apoptosis occurrence appear to reflect prevention of the release of cytochrome c from mito-the initiating event. For example, down-regulation of the chondria, and Akt is unable to inhibit the mechanismcaspase Nedd2 rescues both sympathetic neurons and responsible for cytochrome c release when Bad ser-112PC12 cells from death caused by growth factor withdrawal and ser-136 are mutated to alanines [322]. Bad is de-

21but does not prevent apoptosis induced by down-regulation phosphorylated by the Ca /calmodulin-activated phos-of superoxide dismutase (SOD) [311]. Like caspase 3, phatase calcineurin [323], which is also implicated in NOSNedd2 is expressed in neural precursor cells, is develop- activation.mentally down-regulated [312], efficiently cleaves PARP[313], and its processing from the proenzyme to the active 7.4.3. PKR and phosphorylation of eIF2a

form in response to growth factor withdrawal is inhibited There is recent evidence directly connecting both PKRin a neural cell line overexpressing Bcl-2 [314]. Further activation and eIF2a phosphorylation to apoptosis. Cellsstudies are needed to clarify the identity, activation timing, from PKR knockout mice are resistant to apoptosis in-

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 15

duced by TNF-a, lipopolysaccharides, or viral RNA [331]. FK506 binding to FKBP12 dissociates FKBP12[206,324]. The presence and function of TNFR was not from both receptors and modestly increases their resting

21affected in the knockouts [206] because TNF-a on these probability of being open for Ca transit [332,333], but21cells induced a normal NF-kB response. Unfortunately, after treatment of the RyR with FK506, actual Ca

these studies did not directly examine caspase activation, movement again is only seen at the four discrete ‘subcon-which should have occurred in response to TNF-a unless ductance’ levels [331]. Thus it appears that the overall

21the PKR knockout blocked signaling for caspase transcrip- effect of FK506 on RyR-mediated ER Ca egress is to21tion or activation. However, the issue of whether eIF2a(P) induce a decrease in ligand-activated Ca conductance.

has a direct role in inducing apoptosis has been examined. In recent follow-up of a seminal 1994 study [334],Expression of a mutant eIF2a substituting alanine (cannot FK506 has been shown to both salvage vulnerable neuronsbe phosphorylated) for the ser-51 phosphorylation site after brain ischemia and also to allow them to synthesizeblocks apoptosis induced by TNF-a [324] or PKR [325] stress response proteins within 3 h of reperfusion [335].and also induces down-regulation of Bax expression [326]. This same study examined neuronal death in the hip-Furthermore, expression of a conditional eIF2a mutant pocampus after 7 days reperfusion and found a 16-foldimitating the negative charge at ser-51-P (by substituting increase in the number of surviving neurons in the CA1 (toaspartate for ser-51) induces immediate apoptosis [324], levels statistically no different from neuron density inand activation of caspase 3 [326]. These results suggest sham ischemic controls) of animals treated with FK506.that PKR induces apoptosis through phosphorylation of Similar results with regard to stress protein synthesis andeIF2a. Furthermore, because eIF2a(P) itself appears suffi- neuronal salvage were obtained with FK506 treatment in acient to signal for apoptosis, it is likely that eIF2a kinases model of ischemic stroke induced by 2 h occlusion of aother than PKR are also involved in apoptotic pathways. middle cerebral artery [336].

7.4.4. ER calcium 7.4.5. Immunophilins21As we noted above, depletion of Ca from the ER Immunophilins are a family of cellular proteins that bind

induces PERK (and perhaps PKR) activation, phosphoryla- immunosuppressant drugs, such as FK506, cyclosporin A,tion of eIF2a, and inhibition of protein synthesis, and there and rapamycin. FK506 is a microbially-derived macrolide

21is ER Ca depletion in SVNs during and after transient antifungal antibiotic used clinically to suppress transplant21ischemia [327]. On the ER two Ca channel-regulating rejection [337] and is a structural homologue of rapamycin

receptors are known, the ryanodine and inositol-triphos- but is structurally unrelated to cyclosporin A [338].phate receptors (RyR and IP-3R). Both receptors are Rapamycin and cyclosporin A are not neuroprotectivepresent in vulnerable CA1 neurons, with IP-3R more during post-ischemic reperfusion [334]. FK506 crosses theprominent in these cells and RyR more prominent in the blood–brain barrier, but cyclosporin A does not [339].vulnerable hilar neurons [328]. There is evidence that Both cyclosporin A and FK506 actually inhibit T-cellarachidonate competes with ryanodine for its receptor apoptosis but appear to exert their immunosuppressant[329]; interpreting this result is complicated because, functions by inhibiting T-cell activation, proliferation, andalthough 30 mM arachidonate did not appear to increase stimulation of interleukin-1 production [338].

21the probability that the receptor-gated Ca channel in Generally, cyclosporin A and FK506 bind differentreconstituted ER vesicles was open, arachidonate did in a proteins, but many or all of these proteins catalyze the

45 21dose-dependent fashion stimulate release of Ca . There cis–trans isomerization of peptidyl-prolyl bonds (rotamaseare no published studies of potential interactions between or PPIase activity) [338,340] and thus frequently functionarachidonate and the IP-3 receptor, although IP-3R-de- as peptide folding isomerases in complexes withficient T cells are resistant to apoptosis induced by ionizing chaperones such as heat shock proteins [341]. Because theradiation, FAS ligand, and glucocorticoids [330]. immunophilins have been largely recognized by their

Both the RyR and IP-3R are formed by a tetramer of binding affinity for cyclosporin A or FK506, nomenclaturetheir respective primary proteins, and one 12 kDa peptide, for them is further divided into two large groups,which binds the immunosuppressant tacrolimus (or FK506, cyclophilins and FK-binding proteins (FKBPs).thus FK-Binding Protein — FKBP12), is associated with Several activities of the immunophilins are of substantial

] ] ]each monomer of both receptors [331,332]. Expression of interest with respect to apoptosis. Cyclophilin A (18 kDa),the cloned RyR in naive Spodoptera frugiperda cells B (21 kDa), and C (22.9 kDa) have been found to be(which also do not normally produce FKBP12) generated DNAses independent of their rotamase activity [342]. Thetetrameric RyR receptors, but these receptors did not show DNAse activities of cyclophilin A and B are stimulated by

21 21 1strong ligand activation of Ca conductance and rather increased Ca and inhibited by K (IC 583 mM). This50

exhibited ‘subconductance’ at four distinct levels, sug- DNAse activity is not inhibited by cyclosporin A and isgesting that each monomer included a channel [331]. In directed against double-strand linear, double-strand super-this system, co-expression of FKBP12 generated channels coiled circular, and single-strand linear DNA withthat exhibited ligand-activated opening to full conductance cyclophilin C.B.A [342]. Furthermore, cyclophilin A is

16 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

extensively homologous to an 18 kDa Ca/Mg nuclease nisms emerged after the development of multicelled eu-[343] (NUC18) that may one of the nucleases [344] karyotes.responsible for nucleosomal DNA fragmentation in apop- This suggests that apoptotic mechanisms first arose as atosis. Recent evidence suggests that NUC18 is activated by primitive immune response to viral infection of single cellsproteolysis of an inactive |60 kDa precursor [345]. This in multicelled organisms. Indeed, from the perspective ofproteolytic activation of NUC18 suggests caspase in- the survival of individual organisms to reproduction,volvement, and indeed caspase activation appears to be suicide of the one cell in a one-celled organism does notupstream of endonuclease activation in apoptosis [346]. make any sense. However, suicide of one infected cell in aAnother endonuclease involved in DNA fragmentation multicelled organism could eliminate the replication ma-during apoptosis is DNA Fragmentation Factor 40 (DFF- chinery the virus would utilize to multiply and so promote40, also known as CAD for caspase-activated DNAse). the survival of the multicelled organism. This reasoningThis endonuclease exists in a complex with an inhibitor leads to the idea that apoptotic mechanisms involving(ICAD) that, when cleaved by caspase 3, releases the inhibition of the initiation of protein synthesis may be veryactive DFF-40 [347]. primitive. Apoptosis-naive organisms like yeast regulate

An |21 kDa cyclophilin homologous to cyclophilin D protein synthesis by ser-phosphorylation of eIF-2a in[348] is an integral part of the mitochondrial permeability- response to environmental signals, such as inadequatetransition pore formed in the mitochondrial membrane amino acids. Thus all that had to happen to block viral

21under the simultaneous influence of matrix Ca overload replication was to make the regulation of the introductionand radical stress [349], and both cyclosporin A and of the first amino acid into any new protein responsive to

21 the presence of viral nucleotides, exactly the situation withFK506 inhibit Ca transit of this pore [350]. The integral21 PKR.involvement of an immunophilin with a Ca transiting

21 This perspective also makes intelligible the developmentpore and the ability of FK506 to inhibit Ca transit of theof caspases with very high rates of proteolysis againstpore is consistent with the evidence above suggesting that

21 DNA repair enzymes and the ability to activate nucleases.FK506 inhibits ligand-activated Ca transit across theThe objectives here were to prevent incorporation of theER.viral genome into that of a host cell, to kill the host cell,Some complexes of immunophilins and cyclosporin Aand to degrade viral polynucleotides. Later, governance ofor FK506 inhibit calcineurin (protein phosphatase-2B) butcell survival during embryonic development could havenot other serine / threonine phosphatases [351]. Calcineurinbeen accomplished by signal-transduction regulation ofdephosphorylates and activates Bad [323] and NO-synthet-this platform of apoptosis. By this interpretation, secondaryase [352], and the putative role of NO in formingsignal transduction mechanisms [including the FAS-peroxynitrite radicals together with the inhibition of NOTNFR/FADD-CRADD/caspase 8 activation system, lipid-synthetase by FK506 were the rationale for examining the

21derivative (arachidonate and IP-3) mediated ER Caeffect of FK506 during brain reperfusion [336]. However,shifts, and p-53 regulation of the Bax–Bcl-2 ratio andcomplex formation with calcineurin is not a generalmitochondrial cytochrome c release [356] in response toproperty of FKBPs [353], and derivatives of FK506 that doDNA damage] feed into the more primitive mechanismsnot lead to inhibition of calcineurin are neurotrophic andfor inhibition of protein synthesis, prevention of DNApromote neuron recovery after injury [354]. Furthermore,repair, and DNA degradation.Toung et al. [355] have shown that the neuroprotective

It is clear that a signal for apoptosis can come from anyeffect of FK506 does not involve in vivo inhibition of NOof several organelles (plasmalemma, mitochondria, ER,production during brain ischemia and early reperfusion.and nucleus), and there is evidence for involvement of atleast the ER and mitochondria in activation of apoptotic

7.5. Putting apoptosis together: an evolutionary biochemistry in association with brain ischemia and re-perspective perfusion. From that basic perspective, the complexity of

the details of this biochemistry arises from the presence ofTwo fundamental observations emerge from the above multiple input pathways and the fact that the fundamental

review. First, all multicellular eukaryotic organisms appear execution mechanisms are connected by molecular sig-to have apoptotic systems, but many single-celled eukary- naling.otes are naive with respect to these mechanisms. Second,viruses have developed a variety of mechanisms thatinhibit eukaryotic apoptosis; these mechanisms include at 8. Immediate early genes, growth factor signalleast inhibition of the formation of the membrane re- transduction, and regulation of translation andceptor–adapter linker system for activation of caspases, apoptosisdirect inhibition of caspases, viral expression of Bcl-2, andinhibition of eIF2a kinases. Because single-celled eukary- 8.1. Immediate early genes and ‘heat shock’ proteinsotes do not have these apoptotic mechanisms, theseobservations show that viral resistance to apoptotic mecha- Immediate early genes [357,358] include proto-on-

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 17

cogenes of the c-fos and c-jun families. These proteins stress-activated protein kinases), which are activated inform heterodimers to constitute a variety of AP-1 tran- response to oligomerization of the adapter TRAF-2 toscription factor complexes that bind specifically to consen- either IRE-1 (after activation by ER stress) or TNFR (aftersus promoter sequences upstream of target genes [359] and activation by ligand binding) [383]. There is some evi-also bind TBP (TATA-binding protein), thus facilitating dence that JNK activation and c-JUN phosphorylation areinitiation of transcription [360]. linked to induction of apoptosis [384], but although

In rats [140] and gerbils [141] c-fos and c-jun transcripts Herdegen et al. [385] observed both phenomena after 24 hare elevated in brain homogenates within 30 min of post- brain reperfusion, neither were apparently present at 3 andischemic reperfusion. However, examination of various 5 h reperfusion. Moreover, DNA binding by these proteinscell types during early reperfusion showed that c-fos is post-translationally modified not only by phosphoryla-mRNA is transcribed in large amounts in the damage- tion but also by redox alterations of specific cysteines inresistant dentate granule cells, whereas only modest and the DNA-binding domain [386]; cysteine oxidation (thatdelayed expression is seen in CA and CA pyramidal does not appear to involve disulfide bond formation)1 3

layers of the hippocampus [361–363]. Immunocytoch- blocks DNA binding, and homologous cysteines and redoxemistry has similarly shown early translation of c-FOS regulation are also present in the NF-kB and CREBprotein found in damage-resistant rat cortical layers II and (cyclic-AMP response-element-binding) transcription fac-VI but delayed translation in the SVNs of layers III–V tors [386]. The enzyme (Ref-1) that reduces this cysteine[364] and of the hippocampal CA1 [365]. Furthermore, turns out to be identical to an abasic endonuclease (APE)antisense-mediated inhibition of c-FOS synthesis enhanced that plays a major role in the repair of radical-inducedthe final extent of tissue damage associated with focal DNA damage [386], and the endonuclease and cysteinebrain ischemia [366]. reductase activities of this protein reside in non-overlap-

There are important interrelations between induction of ping domains [387]. Indeed, while the reductase activity ofc-fos and c-jun and the synthesis of other stress proteins Ref-1 /APE is itself down-regulated by oxidation of this[367]. The highly conserved heat shock proteins (HSPs — protein, the endonuclease activity is not [387]. Thisnamed because of their discovery in cells exposed to heat evidence suggests a mechanism down-regulating the activi-stress) are now known to be chaperones to prevent ty of several important transcription factors during radicalaggregation of incompletely-folded peptides. HSPs facili- stress until there is confidence that the genomic codingtate correct folding of proteins [368–370], dissociate sequences are sound.clathrin baskets in the presence of ATP (which is a The issue of the actual transcriptional activity of AP-1presumed requirement for cell membrane recycling) components is further complicated by recent evidence that[371,372], interact with cellular tumor suppressor p53 CHOP, a transcription factor component that forms mainly[373], protect cultured neurons from glutamate toxicity functionally-negative heterodimers with members of the[374], and can be induced by growth factors [375]. Heat C/EBP family and is implicated in apoptosis inductionshock mRNAs [376] and proteins [377,378] are expressed [388], also forms heterodimers with members of the FOS–in the brain during reperfusion. After an ischemic insult, JUN family [389]. Transcription of CHOP occurs inthe SVNs in the hippocampus and cortex transcribe large response to eIF2a(P), is also induced by conditionalquantities of hsp-70 mRNA, but there is little translation of expression of the asp-51 mutant eIF2a imitating a con-HSP-70 by these neurons compared to those in surviving stituitively phosphorylated protein, and is blocked in cellsareas of the hippocampus (i.e. the dentate gyrus) [379]. containing a homozygous mutation for non-phosphor-The c-fos / c-jun /hsp system is directly activated by ioniz- ylatable eIF2a (R. Kaufman, unpublished data). Thus,ing radiation [380], presumably by hydroxyl radical-me- working out the roles of cFOS and cJUN in the response todiated events; the HSP response is induced in the presence ischemia and reperfusion will require at least characteriza-of free radicals [381], but not by increased intracellular tion of (1) regional translation of the messages for thesecalcium alone [382]. proteins, (2) their regulatory phosphorylation state, (3)

The above results suggest the important association of their heterodimerization partners, and (4) their redox state.early transcription and translation of c-fos, c-jun, and In this complicated regulatory environment, although manyhsp-70 with neuronal survival, but the complexity of the of the proteins whose genes are transcriptionally stimulatedmechanisms by which c-FOS and c-JUN are regulated with by AP-1 fall into families that should favor survival [390]regard to DNA binding preclude direct extension of this (antioxidants, growth factors and their receptors, lipidevidence to inferences regarding their transcriptional ac- management, structural proteins, protease regulation, andtivity. The formation of various heterodimers of the FOS– electrolyte and water management), we do not at allJUN family members undoubtedly affects the pattern of understand how c-FOS and c-JUN affect the fate oftranscriptional activation. Furthermore, post-translational neurons after brain ischemia and reperfusion.modifications of these proteins also affect their DNA-binding activity. N-terminal phosphorylation of c-JUN 8.2. Growth factors and neuronal survival(mainly at ser-73) enhances c-JUN activity and is carriedout by a family of kinases (JNKs, also known as SAPKs or Cell growth, proliferation, and differentiation are reg-

18 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

ulated, in part, by growth factors [391–393]. The brain has activated kinases or dephosphorylated by protein phosphat-receptors for several growth factors, including insulin ases that are also activated [418]. It is the modulation of[394–396], fibroblast growth factor (FGF) [397], and the phosphate content of specific proteins that underlies thenerve growth factor (NGF) [398–400], all of which have cellular regulation noted above. Another germane example

21been found in the selectively vulnerable hippocampal and involves the Ca –calmodulin complex, which activatescerebral cortical neurons. Similarly, transforming growth phospholipases and calcineurin; calmodulin is phos-factor beta (TGF-b) [401], insulin-like growth factor-1 phorylated by the insulin receptor [419,420], thereby(IGF-1) [402–406], and epidermal growth factor (EGF) reducing its ability to bind calcium, a reaction that may[407] have been found in the brain or elsewhere in the modulate insulin’s antilipolytic effects. SVNs, includingcentral nervous system. Examples of growth factor re- specifically the cortical pyramids and those in the CA1

sponses following ischemia are an increase in basic FGF in hippocampus, have both insulin receptors and substantialthe rat hippocampus, caudate, putamen, and cortex [408], levels of phosphotyrosine-containing nuclear proteinsincreased production of mRNAs for brain-derived neuro- [421], consistent with transcriptional regulation throughtrophic factor and NGF in rat dentate gyrus and in the rat growth factor tyrosine kinase systems in these cells.hippocampus CA and CA regions [137], and increased1 2

levels of IGF-1 in the lateral cortex, hippocampus, 8.3. Growth factor signal transduction to the apoptoticstriatum, thalamus and pyriform cortex [409]. mechanisms

The mechanisms by which growth factors influence cellsare not completely understood, although we will address Apoptosis is inhibited by a variety of growth factors,the issue of growth-factor signaling to the apoptotic including insulin, IGF-1, NGF, and others. Here we willmechanisms in more detail below. It is clear that trans- focus on signaling from the insulin receptor (Fig. 2) inmembrane receptor proteins for growth factors charac- order to simplify the discussion without intending toteristically include a ligand-activated tyrosine kinase at the suggest that the insulin signaling pathway is unique in itsinner surface of the plasmalemma, and growth factor anti-apoptotic effects, with the possible exception of itseffects are mediated through receptor kinase-induced activation of farnesyltransferase as noted below.‘phosphorylation cascades’ leading to changes in the The anti-apoptotic activity of insulin requires integrityactivity of numerous proteins affecting transcription, trans- of the tyrosine kinase function of the insulin receptorlation, and structural cellular integrity. Thus, it is not [422,423], functional Ras, functional PI-3 kinasesurprising that the administration of growth factors has [424,425], activation of Akt, and activation of the tran-been shown to improve post-ischemic neuronal survival scription factor NF-kB with subsequent increased expres-[410]. Fibroblast growth factor substantially inhibits gluta- sion of TRAF-1, TRAF-2, manganese-superoxide dismut-mate receptor-mediated neuronal damage [411]. Nerve ase (the mitochondrial SOD), cIAP1, and cIAP2 [276,426].growth factor administered either before or after 5 min of Complexes involving the TRAFs and cIAPs inhibit bothcarotid occlusion in mongolian gerbils significantly re- the upstream caspases (caspases 8 and 9) [276]. The classicduced the loss of hippocampal CA neurons [412]. Treat- insulin receptor tyrosine kinase substrates, IRS-1 and IRS-1

ment with IGF-1 after 2 h reperfusion following an 2, may not be involved in insulin’s anti-apoptotic effects.ischemia-hypoxic insult in rats reduced neuronal loss in the Lee-Kwon et al. [422] showed in Chinese hamster ovarycortex [409], and treatment with insulin, either pre- or cells that there was no loss of insulin receptor anti-post-ischemia in the rat, reduced cortical and striatal apoptotic effects with a deletion mutation of the receptor inneuronal necrosis and reduced infarction in the cerebral the juxtamembrane region required for phosphorylation ofcortex, thalamus, and substantia nigra [243,413,414]. IRS-1 and -2. However, these investigators also found that

Insulin effects in particular are complex and affect the insulin-stimulated activation of farnesyltransferase wasfundamental cell regulatory functions well beyond the required for the anti-apoptotic effect of insulin. Rasmanagement of glucose. For example, insulin receptor- farnesylation is required for its attachment to the plas-mediated protein phosphorylation cascades stimulate tran- malemma, but not for activation of the RAS/Raf /ERKscription of c-fos / c-jun, increase mRNA efflux from the pathway.nucleus, induce the enzymatic systems for de novo Several observations indicate a central role for signalinglipogenesis, activate the mitochondrial pyruvate dehydro- through Ras for growth factor-mediated resistance togenase complex, modify the phosphatidylinositol trans- apoptosis. For SVNs, brief periods of ischemic precondi-cytoplasmic signaling system, and stimulate the heat shock tioning are well known to induce substantial resistance toresponse [375,415,416]. The actions of insulin are me- delayed death after a subsequent otherwise lethal transientdiated by a membrane receptor tyrosine kinase that shares ischemic insult. Recent work in primary cultures ofhomology with other growth factor receptors [417]. The cortical neurons has shown that farnesyltransferase in-initial effect of insulin receptor activation is tyrosine hibitors or a dominant-negative Ras block the protectiveautophosphorylation and activation of the intrinsic tyrosine effects of ischemic preconditioning. In contrast, transfec-kinase. Secondarily, other proteins are phosphorylated by tion with a constituitively active Ras provides substantial

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 19

Fig. 2. Many growth factor proteins activate anti-apoptotic signal transduction, which in all cases appears to require Ras and Akt. There is evidence ofunique signaling from the insulin receptor for the C-terminal farnesylation of Ras, which is required for its membrane localization and anti-apoptoticsignaling. The signaling intermediates through which Ras down-regulates PKR are unknown. Other Ras anti-apoptotic signaling involves its directactivation of PI-3 kinase, which generates 39-phosphorylated phosphoinositides that induce up-regulation of p70(s6K) independent of Akt. Phos-phoinositides also bind to pleckstrin homology domains on both phosphoinositide-dependent kinases (PDKs) and Akt. This induces translocation of Akt tothe plasmalemma and activation of Akt’s kinase function through its phosphorylation by PDKs. Growth factor signaling through Akt-mediatedphosphorylation of eIF4E-BP1 and GSK-3, Ras-dependent down-regulation of PKR, and phosphoinositide-dependent up-regulation of p70(s6k) generates acomprehensive enhancement of the mechanisms of translation initiation. Akt phosphorylation (1) of Bad allows Bcl-2 to inhibit Bax-mediated release ofcaspase 9 and cytochrome c from mitochondria, (2) of caspase 9 inhibits its proteolytic activity, (3) of IkB and NF-kB both frees and activates thepro-survival transcription factor NF-kB, and (4) of FKHR1 causes ejection of this pro-apoptotic transcription factor from the nucleus. Mn-SOD inhibitsradical-mediated mitochondrial damage, and complexes of the TRAFs and cIAPs (cellular inhibitors of apoptosis) inhibit caspases.

protection from a subsequent ischemic insult, even without release of cytochrome c and caspase 3 activation [431];2 / 2ischemic preconditioning [427]. Furthermore, the precondi- furthermore, the Rce1 mutation is lethal in mice [429].

tioning effect may also involve NO-dependent Ras activa- We have already seen that Ras signaling down-regulatestion [427] by cys-118 nitrosylation [428]. PKR [210,432], which is involved in both phosphorylation

Ras belongs to a family of proteins that prior to post- of eIF2a and induction of FAS transcription; the signalingtranslational processing have at their C-termini a CAAX pathway from Ras to down-regulation of this eIF2a kinasemotif, in which the cysteine is usually followed by two also appears not to involve Raf /ERK [432]. It is probablyaliphatic amino acids [429]. These proteins undergo three to be expected that Ras signaling will be found to similarlysteps of post-translational processing of their C-termini down-regulate the other eIF2a kinases. Ras-mediated[429] in which (1) the cysteine is modified with a lipid- signaling also up-regulates translation through PI-3 kinase-derived thiofarnesyl or thiogeranylgeranyl group, (2) the dependent modulation of the phosphorylation states ofRce1 endoprotease removes the AAX moiety, and (3) p70(s6k), eIF4E-BP1 [433], and eIF2B [434].methylation then generates the final S-farnesylcysteine PI-3 kinase can be activated by association with IRS-1methyl ester at the C-terminus. In the case of Ras, all three but is also activated by a direct association with Ras and isof these steps are essential to its appropriate localization at an upstream effector for activation of p70(s6k) [435],the plasmalemma, where it produces its effects in response protein phosphatase 1 [436] [dephosphorylates eIF2a(P)],to activation of growth-factor receptors. In this regard, it is Akt [321,437], and NF-kB [438,439]. Over-expression ofvery interesting that insulin (specifically via the insulin PI-3 kinase protects sympathetic neurons from apoptosisreceptor), but not IGF-1, epidermal growth factor, or induced by withdrawal of the insulin-analogue NGF [440].platelet-derived growth factor, induces the phosphorylation Ras can directly activate the PI-3 kinase /Akt pathwayand activation of farnesyltransferase [430]. Thus, insulin independent of the association of PI-3 kinase with IRS-1,appears to have an essential ‘priming’ action for activation and specific mutations on Ras can separate its activation ofof the Ras signaling pathways by other growth factors, and the PI-3 kinase system from activation of the Raf /ERKwe note that farnesyltransferase inhibitors can induce system [441], which are thus seen to be separate signaling

20 B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33

pathways. Active PI-3 kinase principally generates phos- [451] have shown that growth factor up-regulation ofphatidylinositol 3,4 bisphosphate (PI3,4P) and phospha- protein synthesis in neurons involves this system.tidylinositol 3,4,5 trisphosphate (PI3,4,5P) [296], which Thus, anti-apoptotic growth factor signaling involveswe will see are involved in activation of Akt. The PI-3 activation of a Ras /PI-3 kinase /Akt pathway leading to (1)kinase-dependent pathway to activation of p70(s6k) is not general up-regulation of translation initiation systemsclear [442], but activation of this enzyme is abrogated by probably essential for completion of the NF-kB transcrip-expression of a dominant-negative Ras [435]. We note that tional response, (2) maintenance of Bad in its phos-p70(s6k) phosphorylates the ribosomal S6 protein and phorylated state with consequent preserved Bcl-2-mediatedstimulates translation of ribosomal proteins and translation inhibition of Bax-induced channels in the ER and mito-elongation factors whose transcripts contain chondria, (3) Akt-mediated inhibitory phosphorylation of59oligopyramidine tracts [433]. caspase-9, (4) Akt-mediated inhibitory phosphorylation of

The three Akt isoforms are the protein products of the pro-apoptotic transcription factors, and (5) up-regulation ofcellular homolog of the transforming oncogene from the transcription by NF-kB for proteins that enhance mito-AKT8 retrovirus [443]. PI-3 kinase products bind to a chondrial radical resistance and repress the activity of bothpleckstrin homology domain near the Akt N-terminus and upstream caspases. This is a comprehensive and powerfulcause translocation of Akt to the plasmalemma [296], anti-apoptotic system, and priming of this pathway bywhere it is activated by phosphorylation of thr-308 and insulin-induced activation of farnesyltransferase may beser-473 by PI-3 kinase-dependent kinases (PDKs) that essential for anti-apoptotic signaling by other growthappear also to be activated by binding phosphorylated factors. The fact that Akt is caspase degraded [452]phosphoinositides in their pleckstrin homology domains suggests that growth factor activation of this system may[296]. have some urgency during early reperfusion.

Active Akt has several important anti-apoptotic func-tions (Fig. 2). As we saw in the discussion of apoptosis,phosphorylation of Bad by Akt prevents Bad from recruit- 9. Therapeutic considerationsing Bcl-2 from Bax and thus prevents Bax from generatingchannels in ER and mitochondrial membranes. Akt also 9.1. Resuscitationinhibits caspase 9 proteolytic activity by phosphorylating iton ser-196, and expression of ala-196 mutant caspase 9 The data regarding the involvement of insulin in surviv-induces Akt-resistant apoptosis [444]. Activated Akt-1 and al signaling for neurons, as well as other cells, raise-2 can undergo nuclear translocation and phosphorylate the concern about the use of large doses of epinephrine inpro-apoptotic transcription factor FKHR1 [445], thus in- resuscitation of patients from cardiac arrest. Catechol-ducing nuclear export of FKHR1 to the cytoplasm, where amines, such as epinephrine, cause prompt and prolongedit is sequestered by 14-3-3 proteins [446]. Akt also inhibition of insulin secretion by the pancreas [453],phosphorylates and activates IKKa [447] (one of the activate cAMP-induced phosphorylation of the insulinkinases that phosphorylates IkB to induce release of NF- receptor thereby reducing its tyrosine kinase activity [454],kB) and is further involved in anti-apoptotic transactiva- and promote iron-mediated lipid peroxidation [455,456]tion of NF-kB-dependent transcription through phos- and neuronal death [457]. Further examination of thephorylation of the NF-kB p65 subunit [448]. We note that effects of high catecholamine levels on brain growth factorAPAF1 contains a consensus sequence for Akt phosphoryl- levels and receptor responsiveness needs to be conductedation [296] and could also be counter-regulated by this in the setting of cardiac arrest and resuscitation.enzyme. Akt also up-regulates translation by increasing theavailability of eIF4E and inducing activation of eIF2B. As 9.2. Summary of molecular injury mechanismsreviewed above in the discussion of translation initiation,

7eIF4E binds m G-capped mRNAs for delivery to the Brain ischemia (Fig. 1) results in rapid loss of high-ribosome initiation complex. There is a cytoplasmic eIF4E energy phosphate compounds and generalized depolariza-binding protein (eIF4E-BP1 or PHAS-1) that, when de- tion, and adenosine degradation products have the potentialphosphorylated, binds eIF4E and thus inhibits translation to induce the dissociation of GCN2 from HSP 90 withof capped messages. The PI-3 kinase [449]–Akt [442] consequent activation of this eIF2a kinase. SVNs aresystem induces phosphorylation of eIF4E-BP1 and en- characterized by their dendritic fields receiving largehanced translation of capped messages. Akt also induces a amounts of glutamate released by the general depolariza-more general enhancement of translation through up-regu- tion during ischemia. Under these circumstances, bothlation of eIF2B, which regenerates the eIF2-GTP complex voltage-dependent calcium channels and glutamate-reg-

21required for methionine initiation of all new peptides. ulated calcium channels allow a large increase in [Ca ],i21Glycogen synthase kinase-3 (GSK-3) phosphorylates and and this is accompanied by depletion of Ca from the ER

21inhibits not only glycogen synthase but also eIF2B [434]. lumen. These compartmental Ca shifts are associatedAkt directly inactivates GSK-3 [450], and Quevedo et al. with activation of m-calpain, calcineurin, and phospholip-

B.C. White et al. / Journal of the Neurological Sciences 179 (2000) 1 –33 21

ases with consequent proteolysis of calpain substrates peroxidation progress unimpeded while awaiting full ex-(including eIF4G), activation of NOS and potentially of pression of the molecular response to growth factorBad, and accumulation of free arachidonic acid. signaling. Indeed, radical stress can itself induce a 90%

Early during reperfusion, oxidative metabolism of arach- reduction in insulin-induced phosphorylation of ser-473 onidonate is associated with a burst of excess oxygen radical Akt [458].production, iron is released from storage proteins by These considerations argue for a multi-drug therapeuticsuperoxide-mediated reduction, and NO is generated by approach that will probably have to include (1) peptideNOS. These events result in peroxynitrite generation, growth factors for early induction of survival signal-trans-inappropriate protein nitrosylation, and lipid peroxidation, duction and recovery of translational competence, (2)which is substantially localized to the SVNs and in inhibition of calpain, (3) inhibition of radical damage, andultrastructural studies appears to principally damage the (4) caspase inhibition. Rigorous studies to establish suchplasmalemma. The initial recovery of ATP supports very an approach will require evaluation of neurological andrapid phosphorylation of eIF2a, and substantially in- histopathological outcome as well as of molecular end-creased levels of eIF2a(P) persist in SVNs for at least points associated with each intervention component toseveral hours. High catecholamine levels induced by the establish the necessary role of each drug. Such a multi-ischemic episode itself and by catecholamine administra- factorial therapeutic approach to the molecular injurytion down-regulate insulin secretion and induce inhibition mechanisms now appears essential for the development ofof growth-factor receptor tyrosine kinase activity, and substantially improved clinical treatment for brain is-these effects are expected to be associated with down- chemia and reperfusion.regulation of survival signal-transduction through the Raspathway. Release of mitochondrial cytochrome c andcaspase activation occur during the early hours of reperfu- Acknowledgementssion. Thus the SVNs find themselves with substantialmembrane damage, calpain-mediated proteolytic degrada- Supported in part by National Institute of Health Grantstion of eIF4G and cytoskeletal proteins, altered translation NS33196 (Drs. Krause, White, DeGracia, Grossman, andinitiation mechanisms that substantially reduce total pro- Rafols), NS02008 (Dr. Sullivan), NS01832 (Dr. Neumar),tein synthesis and impose major alterations in message GM48517 (Dr. Grossman), and NS39860 (Dr. Rafols). Dr.selection, down-regulated survival signal-transduction, and DeGracia is also supported in part by a grant from thecaspase activation. These circumstances predict indepen- American Heart Association, and Dr. O’Neil is supporteddently fatal pathways involving loss of membrane integrity in part by a grant from the Emergency Medicine Founda-for partitioning ions, progressive proteolysis by calpain tion.and caspases, and inability to check these processesbecause of loss of general translation competence andsurvival signal-transduction.

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