JOURNAL OF NEUROTRAUMAVolume 23, Number 3/4, 2006© Mary Ann Liebert, Inc.Pp. 335–344

Inflammatory and Apoptotic Signaling after Spinal Cord Injury

ROBERT W. KEANE,1,2 ANGELA R. DAVIS,1 and W. DALTON DIETRICH2,3

ABSTRACT

Central nervous system (CNS) destruction in spinal cord injury (SCI) is caused by a complex seriesof cellular and molecular events. Recent studies have concentrated on signaling by receptors in thetumor necrosis factor receptor (TNFR) superfamily that mediate diverse biological outcomes rang-ing from inflammation to apoptosis. From the perspective of basic science research, understandinghow receptor signaling mediates these divergent responses is critical in clarifying events underlyingirreversible cell injury in clinically relevant models of SCI. From a clinical perspective, this workalso provides novel targets for the development of therapeutic agents that have the potential to pro-tect the spinal cord from irreversible damage and promote functional recovery. In this review, wediscuss how the formation of alternate signaling complexes and receptor membrane localization af-ter SCI can influence life and death decisions of cells stimulated through two members of the TNFRsuperfamily, Fas/CD95 and TNFR1.

Key words: alternate signaling complexes; neuron cell death pathways; spinal cord injury; tumor necro-sis factor receptor

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1Department of Physiology and Biophysics, 2The Miami Project to Cure Paralysis, and 3Neurological Surgery, University ofMiami Miller School of Medicine, Miami, Florida.

INTRODUCTION

THE PATHOPHYSIOLOGY of acute spinal cord injury(SCI) is characterized by the shearing of cell mem-

branes and axons, disruption of the blood–spinal cordbarrier, cell death, immune cell transmigration, andmyelin degradation (Blight, 1985; Bethea, 2000; Wax-man, 1992; Beattie et al., 2002; Bethea and Dietrich,2002; Popovich et al., 2002). Deleterious factors suchas pro-inflammatory cytokines, proteases up-regulatedby immune cells and toxic metabolites, and neurotrans-mitters released from lysed cells can induce further tis-sue damage (Benveniste, 1992; Lee et al., 2000; Pearse

et al., 2004). These molecules can also stimulate an in-flammatory reaction, with the subsequent release ofneurotoxic molecules (Blight, 1985; Dusart andSchwab, 1994; Wang et al., 1996; Popovich et al.,2001). This subsequent damage, termed the “secondaryinjury,” causes neuronal cell death and progressive ax-onal loss over time (days to weeks) laterally and longi-tudinally to areas undamaged by the initial trauma(Schwab and Bartholdi, 1996; Crowe et al., 1997; Keaneet al., 2001; Beattie et al., 2002). A primary goal ofspinal cord research has been to prevent or limit sec-ondary cell death that produces further axonal degener-ation and creates a significant barrier to the regenera-

tion of descending and ascending fibers (Banik et al.,1997; Bethea et al., 1999; Popovich et al., 1999).

DELETERIOUS INFLAMMATORYRESPONSES AFTER SCI

Central nervous system (CNS) inflammatory re-sponses that occur after SCI are initiated by peripher-ally derived immune cells (macrophages, neutrophils,and T-cells), and activated glial cells (astrocytes and mi-croglia) that proliferate or migrate into the lesion sitefollowing injury (Popovich et al., 1997; Chatzipanteliet al., 2000; Bethea and Dietrich, 2002; Schnell et al.,1999). T-cells are essential for activating macrophagesand mounting a cellular or immune response. Macro-phages and neutrophils have also been proposed to par-ticipate in tissue destruction and enlargement of the le-sion (Taoka et al., 1997; Popovich et al., 1997, 1999,2001, 2002; Chatzipantelli et al., 2002; Gris et al.,2004). Macrophages and microglia contribute to the sec-ondary pathological and inflammatory response, in partthrough the release of cytokines, tumor necrosis factor(TNF), interleukin-1 (IL-1), IL-6, and IL-10, interferon(Bartholdi and Schwab, 1997), and activation of inter-leukin receptors (IL-4R and IL-2R) (Bethea et al., 1998;Xu et al., 1998; Pan et al., 1999; Carmel et al., 2001;Song et al., 2001; Nesic et al., 2002). Cytokines facili-tate CNS inflammatory responses by inducing expres-sion of additional cytokines, chemokines, nitric oxide(NO), and reactive oxygen (Xu et al., 2001; Pearse etal., 2003). Since inflammation contributes to both con-structive and neurodestructive processes, a more thor-ough understanding of the autoimmune events that oc-cur following SCI may allow us to develop strategiesthat will harness the beneficial effects of inflammationand, hopefully, help to promote functional recovery(Popovich et al., 2001).

Modulation of Inflammation as PossibleTreatment

Prevention of production of inhibitory proinflam-matory molecules by activated mononuclear phago-cytes has been demonstrated to be neuroprotective(Popovich et al., 2002; Gris et al., 2004). Variousstrategies including drug delivery as well as mild hy-pothermia (Martinez-Arizala and Green, 1992; Chatzi-panteli et al., 1999, 2000; Yu et al., 2000) have beenshown to reduce the inflammatory cascade after SCIand provide neuroprotection and improvement in func-tional outcome. Another strategy has concentrated ontargeting selectins on the surface of endothelial or in-flammatory cells (Hamada et al., 1996; Farooque et al.,

1999). Interactions of endothelial cell-adhesion mole-cules with integrins on the white blood cell surfacehave been shown to promote leukocyte extravasationthrough the blood–spinal cord barrier and movementinto the injured spinal cord. In a recent study by Griset al. (2004), a monoclonal antibody to the CD11d sub-unit of the CD11d/CD18 integrin was reported to de-crease infiltration of neutrophils, delayed entry ofhematogenous monocytes macrophages, and lead toneuroprotection and improved neurological outcome ina model of SCI. Thus, continued investigations into themechanisms underlying the activation of inflammatorycascades after SCI could lead to new strategies to in-hibit secondary injury and thus to promote recovery inspinal cord injured patients. Finally, it should be em-phasized that aberrant immune activation that occursfollowing SCI also occurs in many other neurologicaldisorders (Waxman, 1992). Thus, the identification oftargets for the inflammatory cascades will have addi-tional wide therapeutic application for many diseasesbeyond SCI.

CELL DEATH AFTER SCI

After SCI, some cells at the lesion site die by post-traumatic necrosis, whereas others die by apoptosis(Crowe et al., 1997; Shuman et al., 1997; Emery et al.,1998; Springer et al., 1999; Keane et al., 2001; Wardenet al., 2001; Beattie et al., 2002). Spinal cord trauma leadsto increased expression of death receptors and their lig-ands as well as activation of caspases and calpain (Baniket al., 1997; Springer et al., 1999; Casha et al., 2001;Keane et al., 2001). However, there are conflicting re-ports as to the role of cell death in SCI that probably re-flect the known capacity of TNF to be both pro- and anti-apoptotic.

A solution to this paradox has been proposed in the re-cent findings that tumor necrosis factor receptor (TNFR)submembrane localization and the formation of alternatesignaling complexes can alter the fate of cells stimulatedthrough TNFRs. Since death receptor signaling is com-plex, we focused on how distinct signaling complexescan control fate decisions in signaling by two membersof the TNFR family, Fas/CD95 and TNFR1. A better un-derstanding of these processes after SCI will lead to noveltherapeutic interventions to target the acute injury in hu-man SCI.

TNFR Family Signal Transduction after SCI

The TNFR superfamily mediates a wide spectrum ofimportant cellular functions ranging from acute inflam-mation and lymphocyte co-stimulation (Bodmer et al.,

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2002) to apoptosis and other forms of programmed celldeath. The divergent cellular signaling responses oches-trated by these receptors are dependent on cell-type andenvironmental factors (Muppidi et al., 2004). In most in-stances, TNFR1 triggers cellular activation via NF-�B.However, when new protein synthesis is inhibited priorto TNF stimulation, TNFR1 can initiate apoptosis by ac-tivation of apical caspases (Varfolomeev and Ashkenazi,2004). In contrast, Fas/CD95 primarily induces apopto-sis and is a relatively weak inducer of NF-�B. Recent ex-perimental evidence has provided information about howreceptor submembrane localization and the formation ofalternative signaling complexes by two members of theTNFR family—TNFR1 and Fas—can alter the fates ofcells (Muppidi et al., 2004). Here, we discuss how pro-grammed cell death after CNS trauma is a tightly regu-lated process that can be initiated by activation of spe-cific TNFR family members such as Fas/CD95 andTNFR1 and TNFR2. Deletion of TNFR1, TNFR2 (Kimet al., 2001), or Fas/CD95 (Yoshino et al., 2004), orblocking ligand interactions with different TNFR familymembers (Demjen et al., 2004) has emerged as a clini-cally effective therapy for experimental SCI.

Fas/CD95 SignalingFas (CD95, Apo-1) is a member of the TNFR super-

family and is one of six known death receptors. Fas ex-ists as a 45-kDa, type 1 transmembrane protein with anelongated extracellular domain. This extracellular regioncontains three cysteine-rich domains (CRDs), which aretypical of TNF receptors. CRD 1 is contained in the pre-ligand binding assembly domain (PLAD) and is requiredfor the trimerization of Fas prior to ligand binding, whileCRD 2 and CDR 3 are necessary for ligand binding. Fascontains an intracellular Death Domain (DD) that bindsadaptor proteins and perpetuates the initial apoptotic sig-nal. The typical ligand for Fas is FasL (CD95L, Apo-1L,Cd178, TNFSF6). FasL is a 281–amino acid protein pro-duced as a type 2 transmembrane protein and is highlyrestricted to immune cells and cells of the CNS.

In the nervous system, Fas/CD95 activation can leadto cell death of neurons and glial cells (Raoul et al., 2002;D’Souza et al., 1996), but also to enhanced axonal growth(Desbarats et al., 2003). Although much research efforthas been directed to understanding Fas/CD95-inducedcell death signaling in lymphocytes, the signal pathwaysresponsible for Fas/CD95-induced cell death of CNScells and the unanticipated stimulatory property of Fasare not well understood. However, one report has showna link of Fas/CD95 stimulation to ERK activation duringneurite growth (Desbarats et al., 2003). Fas/CD95 and itsligand FasL/CD95L have been implicated in cell deathafter SCI and may play an important role in pathological

degeneration in vivo. Numerous studies have demon-strated that neurons and glia express Fas and FasL in vitroand in vivo (Choi and Beneveniste, 2004). In the normalCNS, basal expression of Fas is barely detectable that itseems to be nonfunctional (Tan et al., 2001). However,Fas and FasL levels have been reported to be elevated inthe compromised CNS in a variety of neurologic disor-ders (Choi and Benveniste, 2004), including SCI (Li etal., 2000; Casha et al., 2001; Demjen et al., 2004).

Recent experimental evidence has shown that neutral-ization of FasL, but not TNF, significantly decreasedapoptotic cell death after SCI (Demjen et al., 2004). Micepretreated with FasL-specific antibodies were capable ofinitiating active hind-limb movements several weeks af-ter injury. The improvement in locomotor performancewas mirrored by an increase in regenerating fibers andupregulation of growth-associated protein gap-43. Thus,neutralization of FasL appears to promote axonal regen-eration and functional improvement in injured adult an-imals (Demjen et al., 2004). However, a true under-standing of how antibodies to FasL reduce cell death andenhance recovery will require more detailed knowledge.The cellular source and target of the ligand in damagedspinal cord need to be identified, and the links betweenaxonal sprouting and recovery of motor function need tobe established. Moreover, for the behavioral experimentsmice were treated with antibodies immediately before in-jury. Thus, clearly protocols will need to be developedto deliver antibodies to the lesion site at later stages toevaluate this therapeutic approach. Moreover, Yoshino etal. (2004) investigated Fas/CD95-mediated apoptosis af-ter SCI using Fas-deficient mutant mice. Mice lackingFas/FasL showed improved functional recovery, de-creased lesion size, and fewer apoptotic cells in the in-jured cord than control littermates, indicating thatFas/CD95 has a prominent role in apoptotic cell deathfollowing SCI.

The mechanism of Fas/CD95 signaling varies betweencell types (Fig. 1). In “type I” cells, a death-inducing sig-nal complex (DISC), consisting of Fas, the Fas-associ-ated death domain (FADD) protein and caspase-8 andcaspase-10, is easily detected after Fas stimulation, andapoptosis cannot be blocked by overexpression of anti-apoptotic Bcl-2 family members. In “type II” cells, thesesignaling complexes are only weakly detected, and Bcl-2 overexpression blocks Fas-induced apoptosis (Scaffidiet al., 1998). Although cells exhibit differences in the ef-ficiency of early Fas signaling events, the molecular ba-sis of differential signaling by Fas, particularly in theCNS is not known.

Redistribution of Fas in the plasma membrane maybe one possible mechanism for regulating the efficiencyof Fas signaling. Recent evidence has shown that

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redistribution of Fas into specialized lipid domainstermed lipid rafts may account for the clonotypic speci-ficity of stimulated T cell death during the immune re-sponse (Muppidi and Siegel, 2004). In type I cells, aportion of Fas is present constitutively in lipid rafts,while in type II cells, the receptor is excluded from lipidrafts during early signaling. The presence of Fas in lipidrafts allows type I cells to undergo apoptosis in responseto weak bivalent anti-Fas stimulation, which does not

induce apoptosis in type II cells. Thus, modulation ofFas localization in lipid rafts may influence the “com-petency signal” for apoptosis. However, it remains tobe determine whether CNS cells can be categorized attype I or type II cells and whether lipid rafts enhancethe efficiency of signaling in the receptor-associated pri-mary signaling complex after CNS injury. Future stud-ies of Fas/CD95 receptor biology in the CNS shouldyield potential targets for therapy.

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FIG. 1. Fas signaling. (A) In activated type I cells, Fas is recruited to lipid rafts. Within lipid rafts, Fas is engaged by FasL,whereby Fas-associated death domain (FADD) and caspase-8 are recruited to Fas to form the death-inducing signaling complex(DISC). Subsequently, caspase-8 can autoactivate and trigger cell death by cleavage of Bid and activation of effector caspases-3 and -7. (B) In type II cells, the DISC is weakly formed and insufficient to directly activate caspase-3. The apoptogenic acti-vation of mitochondria appears to be the main pathway responsible for cell death.

Inhibition of Fas/CD95 Signaling

Several modes of inhibition of apoptotic signalingthrough Fas/CD95 have been characterized in detail (Pe-ter and Krammer, 2003). These include a soluble formof Fas (Cheng et al., 1994) and a secreted decoy recep-tor that binds and competes for the ligand (Pitti et al.,1998). At the receptor level, adenoviral proteins E3/10.4K-14.5K force internalization and/or degradation ofthe CD95 molecule from the cell surface that leads to lossof apoptotic potency, and to the survival of the cell (Els-ing and Burgert, 1998). In addition, protein kinase C(PKC) has recently been shown to regulate Fas/CD95apoptosis. Activation of PKC protected type II but nottype I cells from Fas/CD95-mediated apoptosis due to re-duced cleavage of Bid (Scaffidi et al., 1999). Thus, PKCmay impart its anti-apoptotic action primarily by inacti-vation of Bcl-2 family members.

The cellular FLIP (FLICE-like inhibitory protein) longform (c-FLIPL), which contains a death effector domain,can act as an inhibitor (Irmler et al., 1997) or promoter(Dohrman et al., 2005) of the Fas/CD95 signaling path-way and has been the subject of much debate (Peter,2004). In addition to c-FLIPL, a protein called lifeguard,has been shown to protect cells from Fas but not fromthe TNF-� death signal (Somia et al., 1999). Lifeguardbinds directly the Fas receptor but not to Fas adaptor pro-teins. Its rat homolog, termed “neural membrane protein35” (NMP 35), is upregulated during development ofspinal motor neurons and is highly expressed throughoutthe adult brain and spinal cord, most prominently in den-drites of several neuronal cell types (Schweiter et al.,2002). Recently, it has been shown that lifeguard ex-pression is mediated by the phosphatidylinositol 3-ki-nase-AKT/protein kinase B pathway (Beier et al., 2005).Lifeguard is highly expressed in rat cerebellar granuleneurons (CGNs), and these cells are resistant to FasL-in-duced apoptosis in vitro (Somia et al., 1999). Reductionof endogenous lifeguard expression by antisense oligonu-cleotides or small interfering RNA resulted in increasedsensitivity of CGNs to FasL-induced death and caspase-8 cleavage, suggesting that lifeguard may play an im-portant role in CGNs resistance to FasL-mediated apop-tosis (Beier et al., 2005). Whether lifeguard plays a rolein resistance to FasL-mediated cell death in other popu-lations of CNS neurons remains to be determined.

TNFR Signaling

Previous studies have disagreed as to the expressionof TNFR1 and TNFR2 expression in the normal and con-tused spinal cord. Yan et al. (2003) report no detectableTNFR1 and TNFR2 immunoreactivity in spinal cord ofsham-operated animals. In contrast, TNFR1 immunore-

activity was demonstrated on cells and afferent fiberswithin the dorsal root ganglia, afferent fibers of the dor-sal root, dorsal root entry zone and within lamina I andII of the dorsal horn, whereas TNFR2 expression was ab-sent in these regions (Holmes et al., 2004). FollowingSCI, TNFR1 and TNFR2 expression is elevated in theinjured spinal cord and localized on neurons, astrocytesand oligodendrocytes (Yan et al., 2003), suggesting thatTNFR1 and TNFR2 may play an important role in thepathogenesis following SCI. Targeted gene deletion ofTNFR1 and TNFR2 have shown that nuclear proteinsfrom injured cords of TNFR1�/� mice have reducedNF-�B binding activity compared with the wild-type con-trols (Kim et al., 2001). The decrease in NF-�B activa-tion was accompanied by a reduction in cIAP-2 expres-sion and an increase in the active form of caspase-3. AfterSCI, the TNFR1�/� mice had greater numbers of apop-totic cells, larger contusion size, and worse functional re-covery than wild type controls. TNFR2�/� mice hadsimilar, although not as pronounced, consequences as theTNFR1-deficient mice. These findings support the ideathat TNFR1 and TNFR2 are beneficial for limiting apop-totic cell death after SCI. In contrast, others have reportedthat inactivation of TNF or its receptor did not improveprognosis (Farooque et al., 2001).

A resolution to these conflicting observations has beenproposed in that TNFR1 signaling involves assembly oftwo molecularly and spatially distinct signaling com-plexes that sequentially activate NF-�B and caspases(Micheau and Tschopp, 2003; Muppidi et al., 2004).Early after TNF binding to TNFR1, a TNFR1 recep-tor–associated complex (complex I) forms and containsTRADD, RIP1, TRAF1, TRAF2, and cIAP-1. ComplexI transduces signals that lead to NF-�B activation throughrecruitment of the I-�B kinase “signalsome” high-mole-cular-weight complex (Poyet et al., 2000; Zhang et al.,2000). TNFR1-mediated apoptosis signaling is inducedin a second step in which TRADD and RIP1 associatewith FADD and caspase-8 to form a cytoplasmic com-plex (complex II) that dissociates from TNFR1. How-ever, when complex I triggers sufficient NF-�B signal-ing, anti-apoptotic gene expression is induced and theactivation of initiator caspases in complex II are inhib-ited. If NF-�B signaling is deficient, complex II trans-duces an apoptotic signal. Thus, early activation of NF-�B by complex I serves as a checkpoint to regulatewhether complex II induces apoptosis at a later time pointafter TNF binding.

Recent data has shed new light on how membraneproximal events control fate decisions in signaling byTNFR1 in the CNS after trauma (Lotocki et al., 2004).The results support a model in which a small amount ofTNFR1 is constitutively expressed in the lipid raft mi-

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crodomains. It has been proposed that lipid rafts serve assignaling platforms for variety of receptors includingTNFR1 and Fas. TNFR1 signaling complexes in the nor-mal CNS contain adaptor molecules TRADD, RIP,TRAF1, TRAF2 and cIAP-1 (Lotocki et al., 2004) (Fig.2). Since the TNFR1-TRADD-RIP-TRAF2 complex ini-tiates the pathway leading to survival (Hsu et al., 1996;Wajant et al., 2003), it is probable that the TNFR1 sig-naling complex in the normal CNS initiates a survivalsignal. Moreover, this signaling complex is devoid ofFADD, cIAP-2 and caspase-8 (Lotocki et al., 2004).

CNS trauma induced rapid translocation of TNFR1 tolipid rafts, altered associations with signaling intermedi-ates, and induced transient activation of NF-�B. RIP andcIAP-1 dissociate from TNFR1, whereas FADD andcIAP-2 increase association with this receptor-signalingcomplex in lipid rafts. Because the TNFR1-TRADD-FADD complex initiates the pathway leading to apopto-sis (Hsu et al., 1996), it is possible that alterations in as-sociation of adaptor molecules in the signaling complexare responsible for the switch in the signal transductionpathway from survival in the normal CNS toward apop-tosis after trauma (Fig. 2). Dissociation of RIP from theTNFR1 signaling complex induced by trauma may ab-late or downregulate the NF-�B pathway and facilitatecell death. Additionally, cIAP-1 and cIAP-2 and TRAF1have been identified as NF-�B target genes (Wang et al.,

1998; Schwenzer et al., 1999). Trauma-induced interfer-ence of the NF-�B pathway may result in altered actionsof the caspase-8 inhibitory TNFR1-TRAF-IAP complexto further promote apoptosis (Wajant et al., 2003). By 30min after CNS trauma, caspase-8 was present in TNFR1signaling complexes, supporting the idea that the associ-ation of FADD with TRADD initiates the apoptotic pro-gram by recruiting caspase-8. Thus, in contrast toTNFR1-mediated signaling in cultured cells, these in vivostudies do not reveal an essential role of complex II inthe regulation of TNF-� responses after CNS trauma, butrather indicate that in both the normal and traumatizedCNS, lipid rafts appear to promote the formation of a re-ceptor-associated signaling complex (complex I) to pro-duce different biological outcomes dictated by thesecomplexes. Moreover, complex I in the traumatized CNSharbors activated caspase-8 by 30 min after insult, indi-cating involvement in downstream signaling cascades.Therefore, the death domain of TRADD may act as a cen-tral platform for the recruitment and activation of FADDafter CNS trauma, leading to subsequent binding of cas-pase-8 triggering their activation. These studies supportrecent evidence that the roles for lipid rafts in Fas andTNFR1 signaling varies between cell types (Muppidi etal., 2004). Thus, TNFR signaling is dependent on celltype and subject to influence of other signaling pathways,genetic and environmental factors.

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FIG. 2. Model of lipid raft–mediated tumor necrosis factor receptor 1 (TNFR1) signaling after central nervous system (CNS)trauma. In normal rat CNS, low levels of TNFR1 are present in lipid rafts, and are in complex with TRADD, TRAF1, TRAF2,RIP, and cIAP-1, and signals survival. Early after trauma, increased levels of TNFR1 recruit into lipid raft microdomains (solidcircles), where they associate with the adaptor protein TRADD, Fas-associated death domain (FADD), TRAF2, TRAF1, andcIAP-2. TNFR1 and TRAF1 are polyubiquitinated (Ubq[n]) in lipid rafts after trauma, which leads to degradation via the pro-teasome pathway. In later stages after injury, RIP and cIAP-1 appear to dissociate from the TNFR1 complex by an unknownmechanism, and this complex signals death by activating caspase-8 (Lotocki et al., 2004).

CONCLUSION

Fas/CD95, TNFR1 and TNFR2 have been mainly char-acterized in the immune system and are primarily in-volved in regulating inflammatory and apoptotic re-sponses. However, these receptors are detectable in othertissues, for example the normal and traumatized CNS,raising the possibility that these receptors and their lig-ands have a role in neurological trauma and disease.There is increasing interest in the role of Fas/CD95/FasLsystem in CNS neurons, since inactivation with neutral-izing antibodies confers neuroprotection in animal mod-els of SCI, stroke, and multiple sclerosis (Demjen et al.,2005; Martin-Villalba et al., 1999; Waldner et al., 1997).However, a true understanding of how antibodies to FasLreduce cell death and enhance functional recovery willrequire more detailed knowledge. For example, the sig-naling pathways initiated by the Fas/CD95 death recep-tor in CNS cells have not been delineated. It is not clearif CNS cells exhibit differences in the efficiency ofFas/CD95 signaling and thus can be categorized as typeI or type II cells. The cellular source and target of theligand in damaged CNS tissues need to be identified, andprotocols need to be developed to deliver antibodies tothe lesion at later stages to clearly evaluate this thera-peutic approach.

Recent experimental evidence has provided informa-tion about how receptor submembrane localization andthe formation of alternative signaling complexes can al-ter the fates of cells in vitro, but whether these principlesapply to signaling mediated by TNFR family membersin the normal CNS and after trauma awaits further ex-perimentation. Thus, activation of these signaling path-ways might become promising therapeutic targets for theacute treatment of neurological trauma and disease.

ACKNOWLEDGMENTS

We would like to thank Dr. George Lotocki for the il-lustration. This work was supported in part by NIH PO1NS 38665.

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Address reprint requests to:Dr. Robert W. Keane

Department of Physiology and BiophysicsUniversity of Miami Miller School of Medicine

1600 NW 10th Ave.Miami, FL 33136

E-mail: rkeane@miami.edu

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