Neuroprotective Signal Transduction: Relevance to Stroke

Neuroscience and Biobehavioral Reviews, Vol. 21, No. 2, pp. 193-206, 1997 Copyright 01997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0149-7634/97 $32.00 + .00

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Neuroprotective Signal Transduction: Relevance to Stroke MARK I? MATTSON Sanders-BrownResearch Center on Aging and Departmentof Anatomy & Neurobiology, Universityof Kentucky, Lexingtonj KY 40536, USA

MATTSON, M. P. Neuroprotective signal transduction: relevance to stroke. NEUROSCI BIOBEHAV REV 21 (2) 193–206,1997.—Studiesof ischemic brain injury in cell culture, animal models, and humans have revealed inter- and intra-cellular signaling pathways that increase resistance to cell degeneration and death. Brain injury induces expression of many different growth factors and cytokines which can protect neurons against insults relevant to the pathogenesis of ischemic brain injury including excitotoxicity, hypoxia, hypoglycemia, acidosis, and pro-oxidants. Neuroprotective signal transduction pathways elicit changes that promote the maintenance of cellular ion homeostasis and/or suppress the accumulation of free radicals. For example: basic fibroblast growth factor suppresses expression of a glutamate receptor protein and induces antioxidant enzymes; tumor necrosis factor induces expression of a Ca2+-bindingprotein and Mnsuperoxide dismutase; and secreted forms of ~-amyloid precursor protein hyperpolarize neurons by activating K+ channels. Transcriptional regulation involves activation of tyrosine phosphorylation cascades and NFkB. Interestingly, similar neuroprotective pathways can be activated by moderate levels of cell “stress” such as that induced by glutamate in cell culture or a brief period of cerebral ischemia in vivo. Novel rapid and delayed intracellular neuroprotective signaling mechanisms are being revealed, such as the regulation of Ca2+influx by actin filaments and the induction of genes by Ca2+and radicals. New therapeutic approaches arising from this research include low molecular weight lipophilic compounds that activate neurotrophic factor signaling pathways and agents that selectively depolymerize actin. O 1997 Elsevier Science Ltd. All rights reserved. AmyIoid precursor protein Antioxidant enzymes Basic fibroblast growth factor Calcium Cerebral ischemia Excitotoxicity Hippocampus Neurotrophic factors Platelet derived growth factor Protease nexin Reactive oxygen species Receptor tyrosine kinase Thrombin Tumor necrosis factor

INTRODUCTION

THE LAST 7 years have been remarkably exciting for cell and molecular biologists studying mechanisms of neuronal cell injury and neuroprotection. Prior to 1989, very little was known about molecular responses of the brain to injury, and there were no reports identifying endogenous neuroprotective factors. It had been shown that brain insults can induce the expression of nerve growth factor (NGF) mRNA (29) and basic fibroblast growth factor (bFGF) protein (22). We then discovered that bFGF and NGF (12,71) were very effective in protecting cultured rat hippocampal and cortical, and human cortical neurons against metabolic/excitotoxic insults. Since then, there have been more than 100 reports of various neurotrophic factors protecting one or more neuronal populations against insults relevant to the pathogenesis of ischemic brain injury (73). Importantly, initial cell culture findings have been extended to in-vivo rodent models of focal and transient global ischemia. For example, bFGF administered centrally or systemically to rats 193

protected hippocampal and cortical neurons against ischemic injury (48,83,88), intracerebroventricular administration of NGF reduced ischemic injury to hippocampal neurons in rats (108), and transforming growth factor-fl (TGF-(.3) protected against focal ischemic injury in mice (91). Perhaps the most remarkable revelation has been the large number of neuroprotective factors identified; a partial list includes bFGF, aFGF, NGF, BDNF, NT-3, NT-4/5;IGF-1, IGF2, TGF(3, TNFrY-,TNF~, sApps, PDGFs, GDNF, protease nexin-1, S-100fJ, and several interleukins (6,l&l4,25,33,47,63,65,67,7l,75,83,88,9l,lll,ll2,ll4,l2 8). The present article makes no attempt to catalogue the rapidly growing multitude of reports demonstrating neuroprotective actions of various growth factors and cytokines (see 67, 73 for a partial catalogue). Rather, this article considers the cellular and molecular events effected by the neurotrophic factors and cytokines that result in resistance to ischemic cell injury. What is emerging from such studies of neuroprotective signal transduction is a common underlying

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theme—neuroprotective signaling mechanisms are designed to stabilize cellular ion homeostasis and suppress accumulation of free radicals. This theme is perhaps not too surprising given the plethora of data implicating dysregulation of Ca2+ homeostasis and generation of free radicals in the pathophysiology of ischemic brain injury (see 37, 60, 122for reviews). Such neuroprotective signaling mechanisms are likely involved in phenomena such as “ischemic preconditioning” in which a low level of insult protects neural cells against a subsequent and more severe insult (57). Indeed, based upon the fact that many neuroprotective factors activate some pathways involved in injury (e.g. Ca2+influx), we conceived the “homeopathic hypothesis” for the mechanism of neurotrophic factor actions (4) and expand upon this hypothesis in the present article. This article is not intended to be a comprehensive review and focusses on work done in the author’s laboratory; the full scope of this research area can be acquired from reference lists in the articles cited herein. FREE RADICALS AND CALCIUM AS CONVERGENCE POINTS IN THE NEURODEGENERATIVE PROCESS IN ISCHEMIC BRAIN INJURY

Before delving into the details of neuroprotective signal transduction mechanisms, it is important to clarify the rationale for the various cell culture paradigms and animal models of ischemicneural injury commonly employed. Two general models of ischemic brain injury are commonly used in adult rodents (32,93). One model involves long-term occlusion of a single vessel (usually the middle cerebral artery) resulting in an infarct in the ipsilateral cortex and subcortical structures as well—the damage in this model most closely resembles stroke in humans. The second model involves transient reduction of blood flow to the entire brain induced by transiently blocking blood flow in the common carotid arteries—the brain damage in this model resembles that seen in humans with cardiac arrest and includes selective degeneration of CA1 pyramidal neurons in the hippocampus. Both forms of ischemic injury result in reduced availability of glucose and oxygen to neural cells. Glucose deprivation and hypoxia (effected by reduced 02 tension or exposure to mitochondrial toxins such as cyanide) are commonly employed in cell culture paradigms of ischemic brain injury. Release of glutamate and activation of glutamate receptors (both NMDA and AMPA/kainate) are known to play an important role in neuronal death in animal models of stroke. Cell culture studies have also shown that neuronal injury induced by glucose deprivation or hypoxia can be attenuated by glutamate receptor antagonists (13,79,99).Therefore, excitotoxic insults in cell culture mimic a key component of ischemic brain injury. Cell culture studies have clearly established an important causal role for Ca2+influx through NMDA receptors and voltage-dependent Ca2+ channels in ischemic neuronal injury. Imaging studies of intracellular free calcium levels ([Ca2+]i)in cultured hippocampal and cortical neurons, using fluorescent Ca2+

MATTSON indicator dyes such as fura-2 and flue-3, have shown that excitatory amino acids, glucose deprivation and hypoxia can induce large sustained elevations of [Ca2+]i (12,13,16,63,75).The elevation of [Ca2+]iinduced by these insults is causally involved in neuronal injury because pharmacological manipulations that suppress the elevation of [Ca2+]i(e.g. NMDA receptor antagonists, Ca2+ channel blockers, and Ca2+ chelators) protect neurons from injury and death (13,118,124).Invivo data also implicate Ca2+in the pathogenesis of ischemic injury to neurons. For example, ischemic and excitotoxic insults induce proteolysis of spectrin and microtubule-associated protein-2, cytoskeletal proteins known to be exquisitely sensitive to Ca2+-induced proteolysis (109,115). In addition, Ca2+chelators and glutamate receptor antagonists reduced ischemicinjury in rats (118). Calcium damages cells by activating proteases such as calpains, promoting inositol phospholipid hydrolysis and release of arachidonic acid, and inducing free radical production through several pathways (60,70). Free radicals constitute a second major mediator of ischemic injury to neurons. It is now well-recognized that several types of free radicals are generated as the result of an ischemic episode in vivo, as well as in cell culture paradigms of ischemic injury. For example, studies have shown that hydroxyl radical production, protein oxidation, and lipid peroxidation occur in ischemic brain tissue in vivo (23,24,37). Moreover, direct measurements of reactive oxygen species in neuronal cells in culture have demonstrated that the metabolic/excitotoxic insults induce the production of superoxide anion, hydrogen peroxide and hydroxyl radical (50,70). Free radicals appear to contribute greatly to ischemic neuronal damage and death because antioxidants can protect neurons against ischemic injury in vivo (36), and can also protect cultured neurons against excitotoxic injury (19). Studies of the effects of oxidative insults on neurons are, therefore, of clear relevance to the pathogenesis of stroke. It is upon this foundation of information concerning mechanisms of ischemic brain injury that the following studies of neuroprotective signal transduction arose. BASIC FIBROBLAST GROWTH FACTOR

Basic FGF is believed to be produced by both neurons and glial cells in the brain. Basic FGF binds heparin, and endogenous heparin sulfate proteoglycans are believed to be required for full receptor activation (116). Levels of bFGF were markedly increased in the penumbral region of the infarct following middle cerebral artery occlusion (56), and levels of bFGF mRNA (but not protein) were transiently increased in the hippocampus of rats following transient global ischemia (20). In cell cultures of embryonic rat hippocampus and neocortex, bFGF promoted long-term survival and outgrowth of neurons (80,120). Pre-treatment of hippocampal cell cultures with 10 rig/ml bFGF resulted in the resistance of neurons to glutamate and NMDA toxicity (69,71). Similarly, bFGF protected

NEUROPROTECTIVESIGNALINGAND STROKE cultured striatal neurons against NMDA toxicity (25). Basic FGF also protected cultured hippocampal and cortical neurons against glucose deprivation-induced injury (12), hypoxia (63), and FeSO1 (hydroxyl radical-mediated) toxicity (128). Administration of bFGF to rats in vivo protected hippocampal, cortical and striatal neurons against ischemic and excitotoxic insults (48,83,88). Several lines of evidence suggest that bFGF can act directly on neurons to increase their resistance to ischemic insults. First, bFGF protects neurons in cultures containing greater than 987. neurons (12). Second, neurons express bFGF receptors (12) and exhibit rapid (second to minute) responses to bFGF including elevation of [Ca2+]i(16) and activation of protein kinase C (116). Finally, bFGF affects the expression of several proteins in cultured hippocampal neurons, and the specific actions are consistent with neuroprotection. Thus, bFGF reduced levels of mRNA and protein for a 71 kDa NMDA receptor protein over time periods of 12-48 h, and [Ca2+]i responses to NMDA and vulnerability to excitotoxicity were decreased in cultures pretreated with bFGF (69). In addition, bFGF increased the expression of the 28 kDa Caz+-bindingprotein calbindin in cultured hippocampal neurons (17). Neurons expressing calbindin were previously shown to be relatively resistant to excitotoxic insults, and were found to exhibit reduced [Ca2+]iresponses to glutamate and Ca2+ionophores (44,72,104).Finally, bFGF increased the expression of two key antioxidant enzymes, Cu/Zn-superoxide dismutase and glutathione reductase (70). Ischemic insults to the brain result in reduced energy availability and ATP depletion in neurons. As expected, glucose deprivation in primary hippocampal and cortical cell cultures resulted in ATP depletion (75). Interestingly, growth factors that protect cultured hippocampal neurons against glucose deprivation-induced injury, including bFGF, did not prevent ATP depletion, and yet maintained neurons viable in the face of depletion of ATP levels (75). Mitochondrial function was maintained in the face of ATP depletion in hippocampal cultures pretreated with bFGF. Data from Ca2+ imaging studies and examination of the effects of preventing Ca2+influx on cell damage indicated that Ca2+ influx was involved in mitochondrial damage induced by glucose deprivation (75). Basic FGF suppressed the elevation of [Caz+]iresulting from glucose deprivation. Ca2+ influx resulting from NMDA receptor activation was also shown to be required for glutamate-induced accumulation of cellular peroxides and oxidative damage to neurons; bFGF suppressed glutamate-induced peroxide accumulation of (70). Collectively, these initial data with bFGF indicated that neurotrophic factors protect neurons against metabolic insults by stabilizing Ca2+homeostasis and suppressing free radical production. Subsequent studies with other neurotrophic factors have shown that these are the two general modes of action of neuroprotective growth factors and cytokines (see below).

195 NEUROTROPHINS

The neurotrophins include NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) (8). Each of these neurotrophins is expressed in the brain in distinct regional and cellular patterns. For example, NGF is produced at high levels by hippocampal neurons and BDNF is produced by essentially all neuronal cell types. Moreover, different populations of neurons are responsive to one or more neurotrophins. For example, basal forebrain cholinergic neurons are responsive to NGF, BDNF, NT-3 and NT-4/5, while substantial nigra dopaminergic neurons are responsive to BDNF but not NGF. High-affinity receptors for the different neurotrophins have been cloned and their ligand-specificity examined. TrkA is the receptor for NGF, trkB is activated by both BDNF and NT-4/5, and trkC is the NT-3 receptor. Each of the trks possesses intrinsic tyrosine kinase activity which is believed to initiate cascades of phosphorylation events involving several intermediate kinases and, ultimately, activation of transcription factors (Fig. 1). In addition to the high-affinity trks, a low-affinity neurotrophin receptor (p75) has been identified. The p75 receptor is widely expressed in neurons and glia. The role of the p75 in biological responses to NGF is not completely clear, although data indicate that it modulates the activation of high-affinity neurotrophin receptors. On the other hand, the homology of this receptor to tumor necrosis factor (TNF) receptors (see below) and the recent demonstration of a possible signal transduction pathway involving sphingomyelin hydrolysis and ceramide release (18), suggest an important signaling role for this receptor. Brain injuries, including ischemia, induce the expression of neurotrophins. For example, transient forebrain ischemia and hypoglycemia coma in rats induced the expression of BDNF and NGF (but not NT-3) mRNAs in the hippocampus (53). Intuitively, it seems likely that the increased neurotrophin levels following injury may limit the extent of the injury and promote recovery. In support of this hypothesis, many laboratories have shown that administration of neurotrophins reduces damage induced by many different insults including excitotoxins, oxidative insults and ischemia (73). In-vitro studies of hippocampal and cortical cells have shown that NGF (12), BDNF (14), NT-3 (14), and NT-4/5 (11) can protect neurons against metabolic and excitotoxic insults. NGF also protected cultured hippocampal neurons against direct oxidative insults (127). The cell data have recently been extended to in-vivo models of ischemic brain injury and other neurodegenerative conditions. For example, intraventricular NGF administration protected hippocampal neurons against ischemic injury in rats (108), and BDNF protected dopaminergic neurons against MPTP toxicity in vivo (114). The signal transduction mechanisms mediating neuroprotective actions of neurotrophins are not well understood, but available data suggest that, as

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with bFGF (above), neurotrophins stabilize cellular Ca2+ homeostasis and enhance antioxidant systems. Elevations of [Ca2+]iinduced by glutamate and glucose deprivation in cultured hippocampal neurons were attenuated in cultures pre-treated with BDNF or NT3 (14). Collazo et al. (17) reported that NT-3 and BDNF increase the expression of calbindin in cultured hippocampal neurons. Beyond the effects on calbindin, neurotrophins are likely to affect expression and/or function of other Ca2+-bindingproteins, ion channels, and ion-motive ATPases. Neurotrophins have been found to protect neurons against a variety of oxidative insults, including exposure to hydrogen peroxide (89), FeSOd (127) and mitochondrial toxins (63). NGF and BDNF suppressed accumulation of hydrogen peroxide in cultured hippocampal neurons (70). Several laboratories have recently shown that neurotrophins can increase the levels of several antioxidant enzymes, including catalase, glutathione peroxidase and superoxide dismutase. For example, Sampath et al. (102) showed that NGF induces the expression of mRNAs for catalase and glutathione peroxidase in cultured PC12 cells, resulting in increased activity of these enzymes and increased resistance to oxidative insults. Presumably, classical components of neurotrophin

Growth

signaling pathways are involved in neuroprotective gene expression, including GTP-binding proteins such as ras, and the mitogen-activated protein (MAP) kinases.

PLATELET-DERIVED GROWTH FACTORS

Platelet-derived growth factors occur as A- and Bchains, and are active only as dimers (either AA or BB homodimers, or the AB heterodimer). Two PDGF receptors have been identified and designated a and P. The (3-receptor binds only PDGF dimers containing a B-chain, whereas the a-receptor binds all combinations of PDGF dimers. PDGF Aand B-chains are expressed in the brain where they are apparently produced by both neurons and glia (103). PDGFs have been shown to affect both neurons and glial cells. For example, PDGF induces the proliferation of oligodendrocytes and astrocytes (87,94), and PDGFs promoted survival of cultured cortical and mesencephalic neurons (84). As with many other growth factors, levels of PDGF are increased following brain injury (45). PDGF ci- and &receptors are both expressed in the adult rat brain

Factors

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TNFs

sAPP

.

MAPK

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FIG. 1. Examples of neuroprotective signal transduction cascades activated by growth factors and cytokines. (Left) Many different growth factors activate receptors (GFR) with intrinsic protein tyrosine kinase (tyrk) activity. Tyrosine phosphorylation results in the association of specific proteins, such as src homologyproteins (SH2), with the receptor and initiates a cascade of phosphorylation events including activation of mitogen-activated protein kinases (MAPK). Transcription factors are ultimately activated in this delayed response pathway. More rapid signaling events are also induced by growth factors including inositol phospholipid (e.g. PIP2) hydrolysis resulting in the release of diacylglycerol (DAG) and inositol trisphosphate (IP3) which effect activation of protein kinase C (PKC) and release of Ca2+ from intracellular stores, respectively. (Center) Tumor necrosis factors (TNFs) bind to the p55 receptor (TNFR) resulting in activation of a sphingomyelinase which releases ceramide from sphingomyelin. Ceramide then (indirectly or directly) activates NFkB by causing dissociation of IkB from the p50/p65transcription factor dimer; the dimer translocates to the nucleus and activates specific kB-responsivegenes. TNFs can also induce production of free radicals which may themselves activate cytoprotective pathways. (Right) Secreted forms of ~-amyloid precursor protein (sAPP) bind to receptors (sAPPR) that may possess intrinsic guanylate cyclase activity. Cyclic GMP (cGMP) produced in response to sAPP activates cGMP-dependent protein kinase (PKG) which, through phosphorylation events, results in activation of K+channels, membrane hyperpolarization and reduced caIcium influx. PKG may also activate NFkB resulting in delayed transcription-mediated neuroprotective responses.

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FIG. 2. Hippocampal neurons express PDGF receptors and respond to PDGFs with increased resistance to oxidative insults. (Upper panels) PDGF &receptor immunoreactivity(peroxidase method) is present in hippocampal neurons of adult mouse brain (left) where immunoreactivityis particularly strong in the dentate granule cells (dg) and CA1 pyramidal neurons. Cultured embryonic hippocampal neurons also express high levels of PDGF p-receptor immunoreactivityin both cell bodies and neurites (right). (Lower panels) Dichlorofluorescein fluorescence (induced by cellular peroxides) in cultured hippocampal neurons 10min following exposure to 10pM FeSOt in a control culture (left) and a culture pretreated for 24h with 100rig/mlPDGFBB (right). Note that FeS04-induced accumulation of peroxides is suppressed in neurons in the culture pretreated with PDGF.

wherein a-receptors appear to be largely confined to cells, whereas &receptors are non-neuronal expressed at high levels in neurons (42,43,92,126). PDGF ~-receptors are expressed in many different including hippocampal neuronal populations, neurons in vivo and in cell culture (15; and see Fig. 2). We have found that pre-treatment of rat hippocampal cell cultures with PDGF-#.A or PDGF-BB increases the resistance of neurons to glucose deprivation-induced injury and iron toxicity (15). Peroxide accumulation induced by glutamate or iron was suppressed in neurons pretreated with PDGFs (Fig. 2). Western blot analysis revealed that each PDGF induced tyrosine phosphorylation of several proteins, including a 180kDa band which likely represents the PDGF receptor proteins, and bands at 43 and 45 kDa which may be MAP kinases. The mechanism whereby PDGFs protect neurons may involve induction of antioxidant enzymes because PDGF-AA increased activity levels of Cu/Zn-superoxide dismutase, catalase and glutathione peroxidase, and PDGF-BB increased

catalase and glutathione peroxidase activities (15). Invivo studies of PDGF actions in ischemic brain injury models remain to be performed. TUMOR NECROSIS FACTORS

TNFa and TNF(3 are members of a cytokine family that includes CD40, CD27 and CD30 ligands (1,76), with TNFci being the most intensively studied family member. TNFa levels are normally very low in brain, but striking 100-1000-fold increases in TNFa levels occur in response to brain injuries including ischemic and excitotoxic insults (77,121). The TNF response to injury is particularly noteworthy as peak levels of protein are reached within 2-4 h following injury, much faster than growth factors such as bFGF or the neurotrophins. Two different TNF receptors have been isolated and cloned, and are designated p55 and p75 (38). TNFa and TNF(3 are equally effective in activating each receptor. The signal transduction mechanism of the p55 receptor has been partially elucidated, whereas the signaling pathway resulting from p75

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3. The mitochondrial toxin malonate induces hippocampal injury and production of the calcium-bindingprotein calbindin D28k in astrocytes. Two micromoles of malonate, a reversible inhibitor of succinate dehydrogenase, were injected stereotaxically into the dorsal hippocampus on the left side. The rat was killed 6 days later, and coronal sections were prepared and immunostained with an antibody to calbindin. ‘Thehippocampus injected with malonate was severely damaged, whereas the contralateral hippocampus was largely undamaged. Numerous calbindin-positive astrocytes were present in subcortical white matter on the side of the brain receiving malonate but not on the contralateral side. The lower micrographs show high magnificationsof the white matter lying below the arrow in the corresponding low-power micrographs (upper), FIG.

receptor activation is unknown. Binding of TNF to the p55 receptor activates a membrane-associated sphingomyelinase which cleaves sphingomyelin and releases ceramide (Fig. 1). Ceramide is believed to activate a kinase which phosphorylates the inhibitory subunit of the transcription factor complex NFkB. NFkB consists of three proteins designated, p50, p65 and IkB. As long as IkB is bound to the p50/p65dimer, the transcription factor is inactive. Signals that activate NFkB induce dissociation of IkB, and the p50/p65dimer translocates to the nucleus and induces transcription of genes containing specific kB binding sites in their enhancer region (54). In some types of mitotic cells, TNFs can damage and kill the cells. However, we have found that TNFs protect cultured hippocampal and cortical neurons against several insults relevant to the pathogenesis of stroke. TNFa and TNF13 protected these neuronal populations against glucose deprivation-induced injury, glutamate toxicity (10), and oxidative insults including exposure to iron and amyloid ~-peptide (3). Moreover, Wilde et al. (125) recently reported that TNFcI attenuates hypoxic/hypoglycemic damage in

organotypic hippocampal slice cultures (124). Measurements of [Ca2+]iand peroxide levels showed that pre-treatment with TNFs suppressed the elevation of [Ca2+]iresulting from exposure to glutamate, and suppressed generation of reactive oxygen species (3,10). The mechanism whereby TNFs stabilize Ca2+ homeostasis and suppress accumulation ~f ROS is not fully understood. However, TNFs induced expression of calbindin (10) and the antioxidant enzyme MnSOD (7a). NFkB appears to mediate neuroprotection by TNFs because TNFs induced NFkB DNA binding activity, and suppression of IkB expression using antisense oligodeoxynucleotides resulted in protection against excitotoxicity and amyloid ~-peptide toxicity (3). Moreover, pre-treatment of hippocampal cultures with C2-ceramide (an agent which directly activates NFkB) resulted in increased resistance of neurons to excitotoxicity (34a). In addition to activation of NFkB via the sphingomyelin-ceramide pathway, reactive oxygen species induced by TNF may activate NFkB (105). TNFs also affect glial cells. TNFs induce the proliferation of astrocytes, and data from some laboratories

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(107) indicate that soluble TNFs can damage and kill oligodendrocytes. During the course of our studies of TNF effects on cultured hippocampal cells, we found that TNFs induced expression of calbindin in astrocytes (64). Expression of calbindin in the astrocytes was correlated with increased resistance of the astrocytes to acidosis and calcium ionophore toxicity. Invivo studies revealed that several brain insults, including the excitotoxin kainate and traumatic injury, can induce expression of calbindin in astrocytes in the corpus collosum and subcortical white matter (64). In addition, injection of the mitochondrial toxin malonate into the hippocampus of adult rats resulted in delayed expression of calbindin in white matter astrocytes (Fig. 3). The possible involvement of injuryinduced TNF in this astrocytic response is currently being investigated. Collectively, the data described above suggest, contrary to popular belief, that TNFs may serve cytoprotective functions in the injured brain. Indeed, the rapid TNF response falls well within the “therapeutic window” described by Ginsberg and Pulsinelli (32), and it will therefore be prudent to examine the possible therapeutic potential of manipulations of TNF signaling pathways in animal ischemia paradigms.

METABOLIZES OF ~-AMYLOID PRECURSOR PROTEIN

Although the molecular biology and pathophysiology of the &amyloid precursor protein (~APP) were first considered in the context of Alzheimer’s disease (106), recent studies of the normal functions of @APP indicate that this protein plays fundamental roles in regulating neuronal survival and plasticity in the brain, and that its biological activities are relevant to the pathophysiology of stroke (61). @APP is a 695-770 amino acid glycoprotein that is believed to exist in cells in a transmembrane configuration with a large Nterminal extracellular segment containing several biologicallyactive domains, and a shorter intracellular C-terminus. 13APP was originally identified as the source of the amyloid (3-peptide(A~), a 40-42 amino acid peptide that forms insoluble aggregates (plaques) in the brains of Alzheimer’s patients. A~ resides at the cell surface with a portion of the peptide buried within the membrane. Studies have shown that ~APP can be enzymatically processed in two major ways. One involves a cleavage within the A~ sequence liberating secreted forms of ~APP (sAPP) from the cell surface and precluding release of amyloidogenic A(3. The other cleavage occurs at the N-terminus of the A~

ca2 + Hypoxia Hypoglycemia Glutamate Iron Thrombin

/\ NMDAR

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Ca-Regulating Proteins Antioxidant Enzymes

FIG. 4. Rapid and delayed neuroprotective pathways induced by elevation of intracellular levels of calcium and free radicals. Calcium influx through NMDA receptors (NMDAR) and voltage-dependent channels (VDCC) induces rapid depolymerization of actin filaments and activation of protein kinases. Actin depolymerizationsuppresses calcium influx through NMDAR and VDCC, and phosphorylation of the ion channels may also limit further calcium influx. In addition, calcium activates nitric oxide synthetase (NOS), and metabolizes of the nitric oxide (NO) generated can reduce activation of the NMDAR. Calcium also activates delayed, transcription-dependent, neuroprotective pathways involving,for example, the cyclic AMP regulatory element binding protein (CREB) and the serum response element (SRE). Free radicals are generated in neurons as the result of exposure to several ischemia-inducedinsults, including hypoxia, hypoglycemia,glutamate, thrombin and iron. Free radicals can rapidly alter the function of ion channels, and can also initiate transcriptional events via, for example, the NFkB transcription factor pathway. Neuroprotective gene products whose expression is affected by calcium and free radicals include calcium-regulatingproteins (e.g. glutamate receptors and calcium-bindingproteins) and antioxidant enzymes (e.g. superoxide dismutases, catalase and glutathione peroxidase).

200 sequence and leaves a potentially amyloidogenic Cterminal fragment in the cell membrane. Recent data indicate that sAPPs normally function as modulators of neuronal excitability and promoters of neuronal survival.13APPsare axonally transported, and sAPPs are apparently released from presynaptic terminals in response to excitatory transmitters and electrical activity (86). We found that sAPPs reduce rest [Ca2+]i and attenuate [Ca2+]iresponses to glutamate in cultured hippocampalneurons (65).These actions of sAPPs were very rapid (seconds to minutes) and the sAPPs were active at concentrations of 10-1000pM, suggesting mediation by specific cell surface receptors. The work of Barger et al. (2) indicates that sAPPs activate a membrane guanylate cyclase resulting in elevation of cyclicGMP levels and activation of cyclicGMP-dependent protein kinase. This signalingpathway appears to mediate the ICaz+]i-loweringactions of sAPPs. Patch clamp studies of Furukawa et al. (28) have shown that sAPPs hyperpolarize cultured hippocampal neurons by activatingK’ channels;this action of sAPPs also appears to be mediated by cyclicGMP. The ability of sAPPs to reduce neuronal excitability has been correlated with the ability of the sAPPs to protect cultured hippocampal neurons against excitotoxicity and metabolic insults (65), and to counteract the dendrite outgrowth-inhibiting actions of glutamate (59). Moreover, infusion of sAPPs into the lateral ventricles of adult rats immediately following20 min of transient global forebrain ischemia conferred protection against ischemic damage to hippocampal CA1 pyramidal neurons (111). In addition to rapidly reducing neuronal excitability, sAPPs may also induce longterm, transcription-dependent, events that protect neurons. The latter possibility is suggested by the observations that pre-treatment of hippocampal cultures with sAPPs for 12–24h protects neurons against glutamate toxicity and oxidative insults to an extent greater than that observed with immediate pretreatment (33,65).Interestingly, we have found that sAPPs activate NFkB (4) and induce expression of antioxidant enzymes (author’s unpublished data) in hippocampal cell cultures. In addition to promoting cell survival, sAPPs may also promote synaptogenesis as suggested by the work of Saitoh and co-workers (97); such an action of sAPPs could also result in enhanced recovery following an ischemic event (7). Because f3APP levels rise rapidly following ischemic brain injury (96), it is reasonable to consider that this injury response represents a neuroprotective mechanism. However, depending upon which enzymatic processing pathway predominates, it is also possible that injury-induced (3APP could lead to deposition of amyloid (3-peptidewith long-term detrimental effects. A better understanding of ~APP and sAPP signaling may reveal novel therapeutic strategies for ischemic brain injury. Although Aj3 has been studied from the perspective of Alzheimer’s disease, recent data suggest its potential involvement in neurodegenerative processes occurring in more acute neurodegenerative conditions, including ischemic brain injury. A13 is normally released from brain cells at low levels, and circulates

MATESON in cerebrospinal fluid at nanomolar concentrations (106). While A~ is initially soluble, with increasing age and more so in Alzheimer’s disease, A~ accumulates as insoluble aggregates in blood vessels and brain parenchyma. Cell culture studies have shown that A~ can be neurotoxic and that the toxicity is related to its ability to form insoluble aggregates ‘withcharacteristic filaments with b-sheet structure (46,61). A(3 can damage and kill neurons by a mechanism involving generation of free radicals (including hydrogen peroxide and lipid peroxidation) and disruption of Ca2+ion homeostasis (33,58,66,74).A novel chemistry of A(3 has been described in which the peptide itself, in the presence of molecular oxygen, generates free radical moieties (39). These radicals apparently propagate to the plasma membrane of neurons, resulting in impairment of ion-motive ATPases and loss of ion homeostasis (58). Other amyloidogenic peptides (e.g. pancreatic amylin and b2-microglobulin) appear to share the radical-generating and cytotoxic properties of A~ (68). In addition to being directly neurotoxic, A(3 can greatly increase neuronal vulnerability to excitotoxicity (61). This “endangering” action of A~ could be particularly important in strokes where an excitotoxic mechanism of neuronal death occurs. Several neurotrophic factors have been reported to protect cultured hippocampal and cortical neurons against A~ toxicity,includingbFGF (69), sAPPs (33) and TNF (3). As with protection against excitotoxic and metabolic insults,the neuroprotective mechanisms involved stabilization of Ca2+homeostasis and suppression of free radical accumulation. A~ may play two important roles in the pathogenesis of stroke. First, its accumulation in blood vessels may contribute to cerebral hemorrhage. Indeed, amyloid deposition in blood vessels is the cause of some inherited forms of stroke, the most notable example being cerebral hemorrhage of the Dutch type (100). While this article has focussed on the role of signal transduction mechanisms that protect neurons, the critical role of vascular injury in the genesis of ischemic events suggests investigations of signaling mechanisms that might protect vascular cells from damage will prove valuable. A second role for A@,in the brain parenchyma, is.also likely. Age is a major risk factor for both stroke and Alzheimer’s disease. Thus, damage to blood vessels and deposition of A~ occur concurrently in most aging individuals and, indeed, there is considerable evidence for a direct relationship between deposition of vascular and parenchymal A(3 (90). As described above, the presence of A~ in the brain parenchyma increases neuronal vulnerability to excitotoxic and metabolic insults. It would, therefore, be expected that ischemic brain injury would be more extensive in individuals with deposits of [email protected], deposition of A~ may influence long-term brain damage in stroke survivors because brain injury results in deposition of A13(101). THROMBIN AND PROTEASE NEXIN-1

Well-known for its critical role in blood coagulation cascades (21), emerging data suggest that thrombin

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influences neuronal survival and plasticity in the brain and that thrombin may play an important role in brain injury in general, and stroke in particular. Thrombin receptors are expressed in neurons and glial cells throughout the brain (122). Thrombin receptors are linked to a GTP-binding protein which induces inositol phospholipid hydrolysis and elevation of [Ca2+]i.In the brain, thrombin may arise from prothrombin which is expressed in neural cells, or in injury conditions such as stroke, blood may be a major source of thrombin. In cultured neural cells, thrombin induces neurite retraction (35), and can cause cell death and increase vulnerability to excitotoxic and metabolic insults, and A@toxicity (112,113).Elevation of [Ca2+]iby thrombin appears to be the mechanism whereby it increases neuronal vulnerability to excitotoxicity (112). In addition, thrombin can promote accumulation of reactive oxygen species in neurons (113). Protease nexin-1 (PN-1) is a potent inhibitor of thrombin that is expressed by glial cells and neurons (95,98,112),and is present at particularly high levels around blood vessels. Levels of PN-1 are increased in response to brain injury (40,119). Cell culture and invivo studies have demonstrated neurotrophic and neurite outgrowth-promoting actions of PN-1. For example, PN-1 promoted neurite outgrowth in neuroblastoma cells (35,78), protected cultured hippocampal neurons against metabolic insults (111), and rescued motorneurons against axotomy-induced death in vivo (41). These actions of PN-1 appear to be mediated by inhibition of endogenous thrombin and, indeed, F“N-l reduced [Ca2+]iand attenuated [Ca2+]iresponses to thrombin in cultured hippocampal neurons (112). Moreover, PN-1 attenuated accumulation of peroxides induced by thrombin and A@,indicating that thrombin can suppress free radical accumulation (113). IMMEDIATE AND DELAYED NEUROPROTECTIVE FEEDBACK MECHANISMS INITIATED BY ELEVATION OF CELLULAR CALCIUM AND FREE RADICALS

To this point, I have focussed on the mechanisms by which intercellular neuroprotective factors act on neurons to increase their resistance to ischemic insults. However, another fascinating area of investigation concerns the direct responses of individual neurons themselves to injurious insults. As described above, excitotoxic, metabolic and oxidative insults result in elevation of [Ca2+]iand accumulation of free radicals in neurons. It is now quite clear that Ca2+and free radicals trigger cascades of signaling events in neurons which result in enhancement of mechanisms that maintain Ca2+homeostasis and suppress free radical accumulation. The protective responses triggered by Ca2+and radicals can be classifiedas rapid and not involvingnew expression of gene products, and delayed and mediated by induction of gene expression (Fig. 4). An example of a rapid neuroprotective feedback mechanism triggered by Ca2+was recently described by Furukawa and co-workers (28). They showed that the actin filament-depolymerizing agent cytochalasin D attenuated, and the actin filament-stabilizing agent jasplakinolide protected cultured hippocampal neurons

201 against excitotoxicity.Calcium imaging studies showed that cytochalasin D suppressed Ca2+influx induced by NMDA and membrane depolarization. Because glutamate induces actin depolymerization, these data suggest that actin filaments are involved in a feedback mechanism to limit further Ca2+influx (Fig. 3). Actin filaments might interact directly with ion channels or, perhaps more likely, actin-binding proteins may serve as intermediaries. Phosphorylation of ion channels, ion pumps and other [Ca2+Ji-regulatingproteins by Ca2+dependent protein kinases are also likely to play roles in rapidly enhancing calcium homeostasis. An example of a rapid neuroprotective feedback mechanism triggered by free radicals was recently described by Lipton and co-workers (55). Nitric oxide is produced in response to a number of potentially injurious stimuli including excitatory amino acids and ischemia. Nitric oxide has the potential to damage neurons by interacting with superoxide anion to form highly destructive peroxynitrite. Metabolizes of nitric oxide can modulate the redox state of the NMDA receptor in such a way that its activation is suppressed and Ca2+ influx reduced. Such redox modulation of proteins involved in regulation of Ca2+ homeostasis is likely to be commonplace. Calcium can trigger delayed neuroprotective signaling events involving induction of transcription factors. Two such pathways have been described by Greenberg and co-workers (31). Calcium influx through voltagedependent channels results in phosphorylation of the transcription factor CREB (cyclic AMP response element-binding protein) and activation of immediate early genes (Fig. 3). The second pathway involves activation of the serum response element (SRE) by a mechanism not yet elucidated. Free radicals can also activate delayed neuroprotective signaling pathways, with the classic example being activation of NFkB. Oxidative insults cause dissociation of IkB, freeing the p50/p65 dimer which then translocates to the nucleus and activates kB responsive genes. Perhaps not surprisingly, the antioxidant enzyme Mn-SOD is among the genes activated by NFkB. EMERGING THEMES IN NEUROPROTECTIVE SIGNAL TRANSDUCTION

We recently proposed a “homeopathic” hypothesis to explain the manner in which neurotrophic factors protect neurons against various insults that elevate levels of Ca2+and free radicals (4). The idea is that neuroprotective growth factors and cytokines induce modest and transient increases in [Ca2+]iand free radicals which, in turn, activate neuroprotective genetic programs. This hypothesis is supported by studies showing that bFGF and NGF induce elevation of [Ca2+]iin neuronal populations that they protect against excitotoxic and metabolic insults (16), and TNFs can induce an “oxidative burst” in some cell types, which triggers production of cytoprotective antioxidant enzymes. A second emerging theme concerns the role of apoptosis or so-called “programmed cell death” in ischemic neural injury. Apoptosis is a form of cell

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death in which the cell dies without spewing its contents into the extracellular milieu (49). It is believed, although not yet established, that apoptosis involves the induction of genetic “death programs” involving production of new gene products that actively kill the cell. Cells dying by apoptosis exhibit several characteristics, including cell shrinkage, cell surface blebbing, nuclear condensation, and DNA fragmentation into uniform size fragments. Several recent studies have provided evidence that neurons can die by apoptosis in cerebral ischemia, particularly neurons in the ischemic penumbra (52,85). In general, the same insults can induce either necrotic (rapid cell death involving lysis) or apoptotic neuronal death, depending upon the severity and duration of the insult. Indeed, excitotoxic neuronal injury can manifest itself tis either necrosis or apoptosis depending upon the severity of the insult. An emerging theme concerning apoptosis is that similar mechanisms underlie both apoptotic and necrotic cell death with Ca2+and free radicals playing central roles. Indeed, oxidative insults and conditions that elevate [Ca2+]ican induce either apoptosis or necrosis. Moreover, antioxidants and agents that suppress elevation of [Ca2+]ican protect neurons in injury paradigms of either apoptotic or necrotic cell death. Thus, there may be little distinction between apoptosis and necrosis from the standpoint of mechanisms of cell death. In support of the latter hypothesis, are data showing that essentially the same neurotrophic factor signal transduction pathways can promote neuronal survival in both apoptotic and necrotic cell injury paradigms. A third emerging theme is the concept of “programmed cell life”, a term which we recently coined in order to emphasize the importance of genetic programs designed to keep neurons alive in the face of adversity (62). Each of the transcription-dependent neuroprotective mechanisms described above for bFGF, neurotrophins, TNFs, etc. falls under the rubric of programmed cell life. I propose that many, if not all, of the paradigms of apoptotic neuronal death described previously involve inactivation of the “life program” (e.g. NGF withdrawal-induced death of sympathetic neurons) or insufficient activation of neuroprotective signaling pathways (too little and/or too late). The ability of the protein synthesis inhibitor cycloheximide to protect neurons against death in several settings, including ischemic brain injury, does not provide convincing evidence that “death genes” are responsible for neuronal death in these paradigms. On the contrary, it is well-known that cycloheximide induces immediate early gene expression and does not completely block protein synthesis. It is therefore possible that cycloheximideinduces “life genes” rather than suppressing production of death gene products. In support of the latter hypothesis, Tortosa et al. (117) recently reported that low doses of cycloheximide (sufficient to induce gene expression without markedly reducing protein synthesis?) protected CA1 neurons against transient global ischemic injury in gerbils, whereas high doses exacerbated the damage. These findings are consistent with my contention that levels of cycloheximide that rescue neurons from

MATTSON “programmed cell death” actually induce the expression of immediate early genes and later neuroprotective genes. A fourth emerging theme in neuroprotective signal transduction is that several different neurotrophic factors can elicit similar neuroprotective responses in the same population of neurons. An excellent example is hippocampal neurons wherein bFGF, BDNF and TNF all induce expression of calbindin (10,17) and antioxidant enzymes (70 and author’s unpublished data). This is intriguing because each of these neuroprotective factors activates a different receptor, and different transcription factors may be involved in ultimate activation of the calbindin and antioxidant enzyme genes. These findings suggest that there exists a fundamental set of neuroprotective genes, and that the products of these genes act by stabilizing Ca2+ homeostasis and suppressing accumulation of free radicals. NOVEL THERAPEUTIC APPROACHES BASED ON NEUROPROTECTIVE SIGNAL TRANSDUCTION

Several studies have shown that ischemic brain injury can be reduced by post-ischemia administration of neuroprotective agents, including neurotrophic factors. The fact that a substantial portion of the neuronal death that occurs in focal and global ischemia is delayed for hours, days or even longer, suggests that effective therapies will be possible. Here, I will focus on approaches designed to activate the various neuroprotective signal transduction pathways described above. One logical approach is to administer growth factors. A complicating problem with this approach concerns access of systemically administered trophic factors to the brain. While recent findings in a rat model of focal cerebral ischemia suggest that peripheral administration of bFGF can significantly reduce infarct volume (S. P. Finklestein, Proceedings of the Nineteenth Princeton Conference; 1995), it is unclear as to whether the effect was due to direct actions on neurons or was the result of vasodilation induced by bFGF. Approaches aimed at enhancing growth factor uptake into the brain are being developed, and preliminary studies are promising. For example, linkage of growth factors to transport proteins such as transferring can facilitate entry into the brain (26). A second approach is to identify and develop compounds that stimulate production of neurotrophic factors in the brain. Nabeshima et al. (82) and Lee et al. (51) have reported on several such agents and demonstrated their efficacy in reducing brain injury induced by a variety of insults, including ischemia in rats. A third approach is to identify and develop compounds that activate neurotrophic factor signal transduction cascades. Leading candidates in the latter category include the bacterial alkaloids K-252a and staurosporine which are low molecular weight lipophilic compounds. In cell culture studies of hippocampaI neurons, we have shown that very low concentrations of these compounds (10-1000 pM) protect neurons against excitotoxic, metabolic and oxidative insults (9,34). As with neurotrophic factors,

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the bacterial alkaloids stimulated protein tyrosine phosphorylation and stabilized calcium homeostasis (9). In-vivo studies have shown that systemic administration of these compounds can protect hippocampal neurons against excitotoxic injury, and ameliorate seizure-induced impairment of visuospatial memory in rats (110). Additional technologies are being developed to replace lost neurons and, in theory, could be useful in promoting long-term recovery of function following stroke. For example, gene transfer methods have been used to express neurotrophic factors in fibroblasts and other cell types, and the results of initial studies in which the genetically engineered cells were transplanted into adult rats are encouraging (30). Another approach is to recruit dormant neural “stem cells” which have been shown to be present in the adult mammalian brain (81). These stem cells can be induced to proliferate and differentiate into neuron-like cells in response to neurotrophic factors such as EGF and bFGF. Additional therapeutic approaches include those aimed at activating rapid neuroprotective feedback mechanisms. For example, data indicating that actin filaments regulate calcium influx led to the demonstration of excitoprotective actions (in cell culture and in vivo) of the actin-depolymerizing agent cytochalasin D (28). Agents that increase levels of cyclic GMP (e.g. phosphodiesterase inhibitors) may also prove beneficial in stroke in light of data from the neuroprotective signal transduction mechanism of sAPPs (see above). Finally, I would like to emphasize an aspect of stroke that is generally neglected in the ischemia field. Namely, the incidence of stroke can be greatly reduced

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by several means, including reducing fat and caloric intake, ceasing smoking,increasing exercise, taking low prophylactic levels of aspirin, etc. There is a rich cellular and molecular biology underlying the benefits of the aforementioned preventative strategies that remains to be explored. The events antecedent to stroke largely center on the vasculature. Interestingly, many of the same growth factor and cytokine signaling pathways that influence neuronal survival also play prominent roles in vascular alterations underlying atherosclerosis. Preventative and therapeutic approaches to reducing the incidence and severity of strokes should therefore certainly include the identification and development of agents that suppress vascular alterations. An example of such an approach is the recent work of Zhang et al. (127) who showed that antibodies to intercellular cell adhesion molecule-1 (ICAM-1), a glycoprotein expressed on vascular endothelial cells that promotes leukocyte adhesion, reduced ischemic neuronal damage following middle cerebral artery occlusion in rats. Clearly, neuroprotective signal transduction approaches should not be limited to studies of neurons, and should target each of the various cellular components in the brain. ACKNOWLEDGEMENTS 1 thank J. Geddes and past and present lab members who contributed greatly to the original research described in this chapter, including: S. W. Barger, A. Bruce, B. Cheng, K. Furukawa, Y. Goodman, R. J. Mark and V. L. Smith-Swintosky. Research in the author’s laboratory was supported by the NIH (NS29001, NS30583 and AG10836), the Alzheimer’s Association and the Metropolitan Life foundation.

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